Compositions and methods for the selective detection of tumor-derived viral dna

ABSTRACT

The present disclosure provides methods and compositions of modified oligonucleotide primer and probe combinations, structurally modified with locked nucleic acids, quenchers, and dyes, effective to detect tumor-derived Human Papilloma Virus (HPV) and tumor-derived Epstein-Barr virus (EBV) and, especially, to distinguish viral DNA derived from tumors from viral DNA derived from infectious viral particles.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation of U.S. patent application Ser. No.17/203,068 filed Feb. 22, 2022, which claims the benefit from U.S.Provisional Application Ser. No. 62/990,438, filed on Mar. 16, 2020, allof which are incorporated herein by their reference in its entirety.

SEQUENCE LISTING

This application contains a sequence listing filed in electronic form asan ASCII.txt file entitled “921404-1050 Sequence Listing_ST25” createdon Apr. 16, 2021 and having 135,168 bytes. The content of the sequencelisting is incorporated herein in its entirety.

FIELD

This disclosure relates to detecting viral nucleic acids in bodilyfluids for the purpose of cancer or pre-cancer detection.

BACKGROUND

Viruses are known to promote the development of cancers. For example,infection with high-risk strains of Human Papillomavirus (HPV) isassociated with cancers of the cervix, head/neck, anus, vulva, or penis.Other examples of viruses associated with cancer development includeHepatitis B Virus (HBV), Hepatitis C Virus (HCV), Human T-LymphotropicVirus 1 (HTLV1), Epstein-Barr Virus (EBV), Cytomegalovirus (CMV), HumanEndogenous Retrovirus type K (HERV-K), Merkel Cell Virus, HumanImmunodeficiency Virus (HIV), and Kaposi's Sarcoma Herpes Virus (KSHV).Since circulating tumor DNA (ctDNA) is released by dying cancer cells,these viral nucleic acids may be detectable in the blood of patientswith the corresponding cancer type.

While there is significant interest in detecting viral nucleic acids inbodily fluids for the purpose of cancer detection, there are two majorchallenges associated with this concept. First, the amount ofcirculating tumor-derived DNA (ctDNA) in blood, urine or saliva isextremely low—often on the order of a few molecules per mL of blood.Second, viral nucleic acids present in the circulation are commonly notderived from tumor-associated viruses, but from virions shed fromnon-tumor tissues. This is established by the finding that DNA sequencesfrom many different types of viruses have been identified in the bloodof healthy volunteers (Moustafa et al., PLoS Pathog. 201713(3):e1006292). Given this finding, the detection of viral sequences inthe circulation is, on its own, insufficient to conclude that a patienthas a cancer associated with that virus, since the detected viralsequence may well be derived from normal, non-tumor tissues within thepatient.

For example, any viral sequence detected in a blood sample could beassociated merely with the viral infection itself and have norelationship to tumor burden in the host. This major confounding factorposes a significant challenge to using circulating viral DNA as aspecific marker for cancer detection. Individuals with detectable viralDNA in the circulation could merely be infected with the virus and nothave a cancer or pre-cancer.

Consistent with this, PCR-based methods have been reported for detectingHPV DNA in the circulation of patients with HPV-associated preneoplasiaor cancers, but these methods also detect HPV DNA in the blood of asubstantial proportion of patients who do not have a diagnosis of HPV+cancer (Bodaghi et al., J Clin Microbiol. 2005 43(11):5428-5434; Cocuzzaet al., PLoS One. 2017 12(11):e0188592; Ferreira et al., Pathol ResPract. 2017 213(7):759-765; Chen et al., J Med Virol. 200981(10):1792-1796). Thus, since the presence of HPV virus in thecirculation of a person could be due to infection of normal tissues, itsdetection does not necessarily indicate that a person has anHPV-associated cancer. Because currently available PCR detection methodsare unable to distinguish between tumor-derived HPV DNA and HPV DNAderiving from normal tissue infected with the virus, they lacksufficient specificity to allow early detection of HPV-associatedcancers.

For example, PCR-based methods for HPV detection have demonstrated asensitivity of only about 54% to detect patients with HPV-associatedcancers, which is insufficient to be clinically useful to distinguishpatients who need further evaluation from those who do not need furtherevaluation (Jensen et al., Clin Otolaryngol. 2018 May 15; Higginson etal., Int J Radiat Oncol Biol Phys. 2015 93(3):S78-S-79; Cao et al., IntJ Radiat Oncol Biol Phys. 2012 82(3):e351-358; Dahlstrom et al., Cancer.2015 121(19):3455-3464).

SUMMARY

The present disclosure provides methods and compositions of DNAoligonucleotide primer and probe combinations, structurally modifiedwith locked nucleic acids, quenchers, and dyes, effective to detecttumor-derived viral, e.g., HPV and EBV, DNA and, especially, todistinguish viral DNA derived from tumors from viral DNA derived frominfectious viral particles.

The DNA amplification methods and structurally modified primer/probecompositions disclosed herein quantitatively detect tumor-derived viralDNA in a sample. The disclosed methods and compositions distinguishbetween viral DNA derived from tumors and viral DNA derived fromnon-tumor sources, e.g., from infectious viral particles. In particular,disclosed herein are methods and compositions of detecting or monitoringa human papilloma virus (HPV)-associated malignancy or Epstein-Barrvirus (EBV)-associated malignancy in a subject, wherein the methodsinvolve detecting a presence or absence of at least one circulatingtumor-derived HPV or EBV DNA of a particular size range in a sample fromthe subject. Compositions and kits for conducting these methods are alsoprovided.

In one aspect, the disclosure provides compositions for detectingtumor-derived Human Papilloma Virus type 16 (HPV16), HPV18, HPV31,HPV33, HPV35, or HPV45 in a sample from a subject, wherein a compositionincludes at least (or consists of) a triad, e.g., three, or can includefour, five, six, seven, eight, nine, ten, or more, of modifiedoligonucleotide primer/probe sets selected from: primer/probe setsnumbered 1 to 18 as shown in Table 14 for HPV16, primer/probe setsnumbered 1 to 18 as shown in Table 15 for HPV18, primer/probe setsnumbered 1 to 17 as shown in Table 16 for HPV31, primer/probe setsnumbered 1 to 15 as shown in Table 17 for HPV33, primer/probe setsnumbered 1 to 16 as shown in Table 18, for HPV35 or primer/probe setsnumbered 1 to 17 as shown in Table 19, for HPV45; wherein a firstprimer/probe set of the triad is configured to produce a first ampliconsignal, wherein a second primer/probe set of the triad is configured toproduce a second amplicon signal, wherein a third primer/probe set ofthe triad is configured to produce a third amplicon signal, and whereinthe primer/probe set of the triad with the smallest set numbercorresponds to the first primer/probe set and the primer/probe set ofthe triad with the largest set number corresponds to the thirdprimer/probe set.

For example, if one selects three primer/probe sets numbered 3, 5, and 7from a table, e.g., Table 14, then set 3 is the “first primer/probeset,” set 5 is the “second primer/probe set,” and 7 is the “thirdprimer/probe set.” The smallest set number always corresponds to thefirst set and the largest set number always corresponds to the thirdset, and so the middle number always corresponds to the second set.

In these compositions, the triad of modified oligonucleotideprimer/probe sets can be selected from primer/probe sets numbered 1 to18 as shown in Table 14 for HPV16, or primer/probe sets numbered 1 to 18as shown in Table 15 for HPV18, or primer/probe sets numbered 1 to 17 asshown in Table 16 for HPV31, or primer/probe sets numbered 1 to 15 asshown in Table 17 for HPV33, or primer/probe sets numbered 1 to 16 asshown in Table 18 for HPV35, or primer/probe sets numbered 1 to 17 asshown in Table 19 for HPV45.

In some embodiments, the disclosure provides compositions for detectingtumor-derived Human Papilloma Virus type 16 (HPV16) in a sample from asubject, including a triad of modified oligonucleotide primer/probe setsselected from primer/probe sets numbered 1 to 18 as shown in Table 14for HPV16, wherein a first primer/probe set of the triad is configuredto produce a first amplicon signal, wherein a second primer/probe set ofthe triad is configured to produce a second amplicon signal, wherein athird primer/probe set of the triad is configured to produce a thirdamplicon signal, and wherein the primer/probe set of the triad with thesmallest set number corresponds to the first primer/probe set and theprimer/probe set of the triad with the largest set number corresponds tothe third primer/probe set.

In another aspect, the disclosure provides compositions for detectingtumor-derived Epstein-Barr virus (EBV) in a sample from a subject,including a triad of modified oligonucleotide primer/probe sets selectedfrom primer/probe sets numbered 1 to 28 as shown in Table 20 for EBV,wherein a first primer/probe set of the triad is configured to produce afirst amplicon signal, wherein a second primer/probe set of the triad isconfigured to produce a second amplicon signal, wherein a thirdprimer/probe set of the triad is configured to produce a third ampliconsignal, and wherein the primer/probe set of the triad with the smallestset number corresponds to the first primer/probe set and theprimer/probe set of the triad with the largest set number corresponds tothe third primer/probe set.

In these compositions, the triad can contain three (or four or more)primer/probe sets of which no two primer/probe sets are consecutive, orthe triad can contain three (or four or more) non-consecutiveprimer/probe sets.

In certain embodiments, the primer/probe sets are numbered 3, 5, and 7or 4, 7, and 10, as shown in Table 14 for HPV16, or 4, 7, and 10 asshown in Table 15 for HPV18, or 4, 7, and 10 as shown in Table 16 forHPV31, or 4, 7, and 10 as shown in Table 17 for HPV33, or 4, 7, and 10as shown in Table 18, for HPV35, or 4, 7, and 10 as shown in Table 19,for HPV45. In some embodiments, the primer/probe sets are numbered 4, 5,and 6 as shown in Table 20 for EPV.

In various embodiments, the compositions described herein can furtherinclude one or more reporter moieties, such as reporter dyes, e.g., FAM,HEX, VIC, Cy5™, or Cy5.5. For example, the detection probe 5 of Table 14can be conjugated to reporter moiety HEX and detection probes 3 and 7 ofTable 14 can be conjugated to reporter moiety FAM. In another example,detection probe 7 of Table 14 can be conjugated to reporter moiety FAMand detection probes 4 and 10 of Table 14 can be conjugated to reportermoiety HEX.

In certain embodiments, the composition includes primer/probe setsnumbered 1, 3, and 5, or sets numbered 1, 3, and 8, or probe setsnumbered 4, 6, and 9, or sets numbered 14, 16, and 18, or sets numbered1, 2, and 5, or sets numbered 10, 12, and 13, as shown in Table 14 forHPV16.

In another aspect, the disclosure provides methods for detectingtumor-derived Human Papilloma Virus type 16 (HPV16), HPV18, HPV31,HPV33, HPV35, or HPV45 in a sample from a subject, the methods includingproviding triads of any of the modified oligonucleotide primer/probesets of any of the compositions described herein as recited in Tables14, 15, 16, 17, 18, and 19; fractionating a plurality of HPV DNAfragments from the sample into droplets at a concentration wherein only0 or 1 molecule of the DNA fragments is present in each droplet;amplifying HPV DNA in each droplet with the triad of primer/probe setsto produce amplicon signals; and detecting in each droplet any ampliconsignals; wherein detection within a droplet of the second amplicon, butnot the first or third amplicon, indicates that the HPV DNA fragmentfractionated into the droplet is a tumor-derived HPV DNA fragment.

In these methods, the triad can contain three primer/probe sets fromTable 14 and the method detects tumor-derived HPV16, or the triad cancontain three primer/probe sets from Table 15 and the method detectstumor-derived HPV18, or the triad can contain three primer/probe setsfrom Table 16 and the method detects tumor-derived HPV31, or the triadcan contain three primer/probe sets from Table 17 and the method detectstumor-derived HPV33, or the triad can contain three primer/probe setsfrom Table 18 and the method detects tumor-derived HPV35, or the triadcan contain three primer/probe sets from Table 19 and the method detectstumor-derived HPV45.

In another aspect, the disclosure provides methods for detectingtumor-derived Human Papilloma Virus type 16 (HPV16) in a sample from asubject, the method including providing a triad of modifiedoligonucleotide primer/probe sets of any of the composition describedherein as recited in Table 14; fractionating a plurality of HPV DNAfragments from the sample into droplets at a concentration wherein only0 or 1 molecule of the DNA fragments is present in each droplet;amplifying HPV DNA in each droplet with the triad of primer/probe setsto produce amplicon signals; and detecting in each droplet any ampliconsignals; wherein detection within a droplet of the second amplicon, butnot the first or third amplicon, indicates that the HPV DNA fragmentfractionated into the droplet is a tumor-derived HPV DNA fragment.

In another aspect, the disclosure provides methods for detectingtumor-derived Epstein-Barr virus (EBV) in a sample from a subject, themethods including providing triads of any of the modifiedoligonucleotide primer/probe sets of any of the compositions describedherein as recited in Table 20; fractionating a plurality of EBV DNAfragments from the sample into droplets at a concentration wherein only0 or 1 molecule of the DNA fragments is present in each droplet;amplifying EBV DNA in each droplet with the triad of primer/probe setsto produce amplicon signals; and detecting in each droplet any ampliconsignals; wherein detection within a droplet of the second amplicon, butnot the first or third amplicon, indicates that the EBV DNA fragmentfractionated into the droplet is a tumor-derived EBV DNA fragment.

In all of the methods described herein, the DNA fragments can befractionated into micro-droplets by emulsification, and/or the DNA canbe amplified using a PCR based method.

In all of the methods described herein, the sample can be a blood,saliva, gargle, or urine sample, e.g., a blood sample.

In other aspects, the disclosure provides methods for detectingtumor-derived HPV16, HPV18, HPV31, HPV33, HPV35, or HPV45, in a samplefrom a subject utilizing a composition of oligonucleotides of primersand probes including any triad of primer/probe sets from Table 14, 15,16, 17, 18, or 19, respectively, wherein a first primer/probe set,consisting of two primers and one probe, is configured to produce afirst amplicon signal, wherein a second primer/probe set, consisting oftwo primers and one probe, is configured to produce a second ampliconsignal, wherein a third primer/probe set, consisting of two primers andone probe, is configured to produce a third amplicon signal;fractionating DNA fragments from a sample from the subject into dropletsat a concentration wherein only 0 or 1 DNA fragments are present in eachdroplet; amplifying the DNA in each droplet with the series ofprimer/probe sets to produce the amplicon signals; and detecting in eachdroplet the number of amplicon signals, wherein detection of the secondamplicon but not the first or third amplicon is an indication that thesubject has tumor-derived HPV16, HPV18, HPV31, HPV33, HPV35, or HPV45.

Also disclosed are methods for detecting tumor-derived EBV in a samplefrom a subject utilizing a composition consisting of non-consecutivetriad of primer/probe sets from Table 20, wherein a first primer/probeset, consisting of two primers and one probe, is configured to produce afirst amplicon signal, wherein a second primer/probe set, consisting oftwo primers and one probe, is configured to produce a second ampliconsignal, wherein a third primer/probe set, consisting of two primers andone probe, is configured to produce a third amplicon signal;fractionating DNA fragments from a sample from the subject into dropletsat a concentration wherein only 0 or 1 DNA fragments are present in eachdroplet; amplifying the DNA in each droplet with the series ofprimer/probe sets to produce the amplicon signals; and detecting in eachdroplet the number of amplicon signals, wherein detection of the secondamplicon but not the first or third amplicon is an indication that thesubject has tumor-derived EBV.

Methods for DNA fragmentation into droplets for digital PCR are known,and include fractionation into micro-droplets by emulsification. In someembodiments, the DNA is fractionated based on size prior to performingemulsification into micro-droplets. This can be done, for example, toisolate a range of DNA fragments for quantification by size.

The DNA fragments can be amplified using any known method, such as a PCRmethod or a non-PCR method.

In some embodiments, the subject has never been diagnosed with orsuffered from an HPV-associated or EBV-associated malignancy. In otherembodiments, the subject has previously undergone treatment for anHPV-associated or EBV-associated malignancy.

The disclosed methods can also be used to monitor treatment. Therefore,also disclosed herein is a method of monitoring HPV-associated orEBV-associated cancer or malignancy in a subject, that involvesquantifying tumor-derived cell-free HPV or EBV viral DNA in bloodsamples collected at two or more time points during treatment of asubject being treated for the HPV-associated or EBV-associatedmalignancy using the disclosed methods. In some of these embodiments,the presence of the tumor-derived cell-free HPV or EBV viral DNA insamples collected at later points in time can be indicative that thesubject being treated for an HPV-associated cancer or malignancy willhave an increased likelihood for recurrence of the HPV-associated orEBV-associated malignancy. Likewise, in some of these embodiments, therapid clearance of or absence of said tumor-derived cell-free HPV or EBVviral DNA in samples collected at later points in time can be indicativethat the subject being treated for the HPV-associated or EBV-associatedmalignancy will have a decreased likelihood for recurrence theHPV-associated or EBV-associated malignancy. In these cases, the methodcan also further involve treating the subject with a reduction inradiation therapy and/or chemotherapy if the subject exhibits a rapidclearance of or an absence of the tumor-derived cell-free DNA sequencesof HPV or EBV in samples collected at later points during the course oftreatment.

According to aspects of the disclosed method, longitudinal analysis oftumor-derived virus nucleic acids in the blood with the disclosed methodmay be utilized for early detection of virus-positive cancers inindividuals who do not present any symptoms related to their malignancyor in whom such symptoms have not yet been identified by clinicians. Asdemonstrated herein, the disclosed method allows previously unachievablelevels of sensitivity and specificity for detecting circulatingtumor-derived viral nucleic acids that is applicable to patients withHPV+ or EBV+ cancers.

In some embodiments, the disclosed methods can be applied to determinethe likelihood of initial diagnosis or recurrence of a virus-associatedcancer comprising detecting the presence or absence of at least onecirculating tumor nucleic acid marker for the relevant virus in bloodsamples collected from a subject at a single time point orlongitudinally over time.

In some embodiments, the disclosed methods can be applied for selectingtreatments for oropharyngeal squamous cell carcinoma (OPSCC) or othervirus-associated cancer comprising detecting the presence or absence ofat least one circulating tumor-derived human papilloma virus (HPV) DNAin samples collected prior to starting treatment and/or at variouspoints in time during treatment from a subject diagnosed with OPSCC orbeing treated for OPSCC, wherein the presence and/or quantity of saidcirculating tumor-derived HPV DNA in samples collected at later pointsin time is indicative that the subject being treated for OPSCC will havean increased likelihood for OPSCC recurrence. Alternatively, rapidclearance of or absence of said circulating tumor-derived HPV DNA insamples collected at later points in time is indicative that the subjectbeing treated for OPSCC will have a decreased likelihood for OPSCCrecurrence, and treating the subject with a reduction in radiationtherapy and/or chemotherapy if the subject exhibits a rapid clearance ofor an absence for said circulating tumor nucleic acid marker for HPV ata specific point in time after initiating cancer therapy.

Also disclosed herein are methods of determining a treatment regimen forhuman papilloma virus (HPV)-associated cancer or malignancy comprisingdetecting the presence or absence of at least one circulatingtumor-derived HPV DNA in samples collected at different points in timeduring treatment from a subject diagnosed with HPV-associated cancer orbeing treated for said HPV-associated malignancy, wherein the absence ofor the rapid clearance of said circulating tumor-derived HPV DNA insamples collected at later points in time during treatment is indicativethat the subject can be treated with a reduction in radiation therapyand/or chemotherapy.

Also disclosed herein are methods of detecting, monitoring and/ortreating a HPV-associated or EBV-associated malignancy in a subject, themethod comprising detecting a presence or absence of at least onecirculating tumor-derived HPV or EBV DNA in samples collected from thesubject at various points in time during a course of treatment, whereinthe presence of the circulating tumor-derived HPV or EBV DNA in samplescollected at later points in time during the course of treatment isindicative that the subject has an HPV-associated or EBV-associatedmalignancy or an increased likelihood for an HPV-associated orEBV-associated malignancy recurrence, and the rapid clearance of orabsence of circulating tumor-derived HPV or EBV DNA in samples collectedat later points in time during the course of treatment is indicativethat the subject does not have an HPV-associated or EBV-associatedmalignancy or has a decreased likelihood for an HPV-associated orEBV-associated malignancy.

Also disclosed herein are methods for monitoring and/or treating aHPV-associated or EBV-associated malignancy in a subject comprisingdetecting levels of a circulating tumor-derived HPV or EBV DNA insamples collected at various points in time from the subject diagnosedwith or being treated for the HPV-associated or EBV-associatedmalignancy; determining a circulating tumor-derived HPV or EBV DNAprofile for the subject; and adjusting a treatment regimen for theHPV-associated or EBV-associated malignancy according to the circulatingtumor-derived HPV or EBV DNA profile, wherein a subject with a favorablecirculating tumor-derived HPV or EBV DNA profile is treated with ade-intensified treatment regimen.

Also disclosed herein are kits including components and compositions asdescribed herein for detecting, monitoring, and/or treating malignanciesin a subject as described herein, and instructions for the use thereof.For example, the kits can contain primer/probe sets set forth in Tables14 to 20, and instructions for the use thereof.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B: FIG. 1A) Dual fragment HPV16 assay to detect tumorviral DNA. Shown are the digital PCR fluorescence detection plots forsimultaneous detection of two distinct fragments of the HPV16 genome.Examples are shown for the single positive controls, dual fragmentpositive control, intact HPV genome control, and two patient plasma DNAsamples. Gates used to quantify single- and double-positive droplets areshown, as described in the methods. FIG. 1B) Analysis of 12 plasma DNAsamples from patients with HPV+ oropharyngeal cancer using this assayuniformly demonstrates the presence of tumor-derived viral DNA—indicatedby abundance of both fragments in single positive droplets and very rareco-occupancy of both targets in the same droplet (in contrast to theintact genome control).

FIG. 2: Analysis of a large cohort of patient blood samples using theHPV blood assay described in Example 1. There was no HPV tumor viral DNAdetected in 30 healthy volunteers and 50 patients with HPV negativecancers (breast and pancreatic). In contrast, 95/102 patients with adiagnosis of HPV positive oropharyngeal cancer have pre-treatmentcirculating tumor HPV DNA in plasma using the blood test described here.The observed number of copies of HPVDNA detected by the assay is alsoindicated, highlighting the dynamic range of the test. Based on thisdata, estimates for assay specificity and sensitivity are 100% and 93%,respectively, at the time of initial diagnosis.

FIGS. 3A-3D: FIG. 3A) Schematic of timepoints when blood was sampled ina cohort of oropharyngeal cancer patients prior to, during, and afterreceiving chemoradiotherapy (CRT). FIG. 3B) Two patterns of plasmactHPV16 profile observed after initiating CRT. In some patients (redlines), ctHPVDNA levels are highest pre-treatment and diminish afterinitiating therapy. In other cases (blue lines), ctHPVDNA levelsincrease soon after starting treatment, possibly due to a spike incancer cell death. In all cases, ctHPVDNA levels are markedly reduced atthe end of CRT, indicating that ctHPVDNA levels are correlated with thevolume of active cancer in patients. FIG. 3C) A subset of patients haverapid clearance kinetics of ctHPVDNA during CRT, with >95% of ctHPVDNAcleared by week 4 of CRT. FIG. 3D) Another subset of patients hasdelayed kinetics of ctHPVDNA clearance after CRT, which may becorrelated with poor or delayed response to CRT.

FIG. 4A-F: A subset of 20 patients with HPV+ OPC had next generationsequencing (NGS) analysis of their primary tumor as well as ctHPVDNAanalyses of their blood to investigate potential correlation betweenthese assays. FIG. 4A) There was a strong correlation between the HPVDNAdPCR assay described in Example 1, when applied to the tumor biopsyspecimen, and tumor HPV copy number assessed by NGS. This validates boththe HPVDNA assay and NGS using orthogonal assays on the same samples.FIG. 4B) There is a statistically significant correlation between tumorHPV copy number per cellular genome and pre-treatment ctHPVDNA in theblood, normalized to tumor volume (“ctHPVDNA density”). This indicatesthat the level of ctHPVDNA detected in blood is correlated with HPV copynumber in the associated tumor. FIG. 4C) A bioinformatics analysispipeline was developed to distinguish HPV+ HPV cancers that haveevidence of HPV integration into the human genome versus those cancersthat have purely episomal HPV, using the tumor NGS data. FIG. 4D) Acircos plot is shown demonstrated the observed rearrangements in acancer with episomal HPV (left) and integrated HPV (right). The examplewith integrated HPV shown here has an integration site that maps to aregion on chromosome 8 (see the dark black lines). FIG. 4E) Higher tumorHPV copy number correlates with a greater likelihood of non-integrated(episomal only) HPV. FIG. 4F) Higher ctHPVDNA levels in blood alsocorrelate with a higher likelihood of non-integrated (episomal only) HPVin the associated cancer. Thus, ctHPVDNA can also provide information onthe status of the HPV genome in the associated cancer—i.e., if it isintegrated versus episomal.

FIG. 5A-B: FIG. 5A) A schematic for stratifying patients based on theirctHPVDNA profile. Patients with abundant pre-treatment ctHPV16DNA thatis rapidly cleared (>95% by day 28) are classified as having a favorablectHPVDNA profile. All other patients are classified as having anunfavorable ctHPVDNA profile. FIG. 5B) Favorable ctHPVDNA profile isobserved in ˜30% of patients with clinically favorable (<T4 and <=10pack-year smoking history) and in ˜30% of patients with clinicallyunfavorable (T4 or >10 pack-year smoking history) disease.

FIG. 6A-B: FIG. 6A) Proportion of patients in each subgroup who had apositive post-treatment neck dissection (i.e., regionally persistentdisease), regional recurrence, and distant metastasis. Patients who hadunfavorable clinical risk factors and an unfavorable ctHPVDNA profilehad the highest risk of adverse disease events. FIG. 6B) Kaplan-Meieranalysis of regional disease (persistent or recurrent) free survivalstratified by clinical risk and ctHPVDNA profiles. Patients with aFavorable ctHPV16DNA Profile had 100% regional disease control,regardless of smoking history (5 patients were heavy smokers). Incontrast, clinical higher risk patients with an Unfavorable ctHPVDNAProfile had significantly reduced regional disease control. P,two-tailed log rank test for a trend.

FIG. 7: The ctHPVDNA test described here was applied to a cohort of 73patients who had completed treatment for HPV+ Oropharyngeal cancer.These patients had no evidence of disease and were clinicallyasymptomatic. They were monitored with the ctHPVDNA blood test at eachfollow up visit. 60 out of 73 patients had undetectable ctHPVDNA at allfollow up visits, and none of these patients developed diseaserecurrence during the follow up period. In contrast, 13 out of 73patients developed a positive ctHPVDNA blood test during the clinicalfollow up period. 9 out of these 13 patients have also developedclinically evident disease recurrence. The ctHPVDNA blood test waspositive up to 6 months prior to identification of recurrent disease ona diagnostic radiology scan. The remaining 4 patients who have apositive blood test are being closely monitored for possible diseaserecurrence.

FIG. 8: Case example from the ctHPVDNA surveillance study. This patientwas clinically asymptomatic and believed to be cancer-free. In June 2017he developed a positive ctHPVDNA blood test. Soon thereafter, he wasexamined by an oncologist and was found to have no evidence of disease.Three months later, he was again examined by a clinician who did notidentify any evidence for disease recurrence. However, the patientreported some neck/shoulder pain, which was believed to bemusculoskeletal in nature. A neck/shoulder MRI was ordered, and on thisexam—4 months after the blood test was positive—an isolated abnormallyenlarged lymph node was identified that was subsequently biopsied andconsistent with recurrent HPV+ oropharyngeal cancer.

FIG. 9: Representative embodiment of method to detect tumor-derived HPVviral DNA assay using the triad primer/probe sets presented in Tables14, 15, 16, 17, 18, 19, and 20. Shown are the digital PCR fluorescencedetection plots for a multiplexed digital PCR reaction to detecttumor-derived HPV16 DNA in a patient blood sample that includes modifiedprimer probe sets 3, 5 and 7 from Table 14, where detection probe 5 isconjugated to HEX and detection probes 3 and 7 are conjugated to FAM.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, usefulmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference in their entireties, as if each individualpublication or patent were specifically and individually indicated to beincorporated by reference, and are incorporated herein by reference todisclose and describe the methods and/or materials in connection withwhich the publications are cited. The citation of any publication is forits disclosure prior to the filing date and should not be construed asan admission that the present disclosure is not entitled to antedatesuch publication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of chemistry, biology, and the like, which arewithin the skill of the art.

The examples are put forth so as to provide those of ordinary skill inthe art with a complete disclosure and description of how to perform themethods and how to make and use the compositions and probes disclosedand claimed herein. Efforts have been made to ensure accuracy withrespect to numbers (e.g., amounts, temperature, etc.), but some errorsand deviations should be accounted for. Unless indicated otherwise,parts are parts by weight, temperature is in ° C., and pressure is at ornear atmospheric. Standard temperature and pressure are defined as 20°C. and 1 atmosphere.

Before the embodiments of the present disclosure are described indetail, it is to be understood that, unless otherwise indicated, thepresent disclosure is not limited to particular materials, reagents,reaction materials, manufacturing processes, or the like, as such canvary. It is also to be understood that the terminology used herein isfor purposes of describing particular embodiments only, and is notintended to be limiting. It is also possible in the present disclosurethat steps can be executed in different sequence where this is logicallypossible.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

As used herein “human papilloma virus” or “HPV” refers to small,non-enveloped, double-stranded DNA viruses that infect the cutaneousand/or mucosal epithelium. As understood by those skilled in the art,over 100 HPV genotypes are known to exist. Sexually transmitted,mucosotropic HPVs are further subcategorized as high risk (e.g. HPV16and HPV18) or low risk (HPV6 and HPV11).

As used herein, HPV-associated malignancies include those of the headand neck (larynx, oral cavity, oropharynx, tonsils, and esophagus),respiratory tissue, breast, skin, cervix, vulva, penis and anus.Malignancy and cancer are used interchangeably.

As used herein, “detecting” or “detection” means testing, screening, orotherwise determining the presence and/or absence of at least one tumornucleic acid marker for HPV in a sample. Such detecting or detection canbe carried out by methods described herein, including those known in theart as applicable to this technology, for example, nucleic acidamplification, hybridization-based detection, microarray, and nextgeneration sequencing.

Also as used herein, the terms “treat,” “treating” or “treatment” mayrefer to any type of action that imparts a modulating effect, which, forexample, can be a beneficial and/or therapeutic effect, to a subjectafflicted with a condition, disorder, disease or illness, including, forexample, improvement in the condition of the subject (e.g., in one ormore symptoms), delay in the progression of the disorder, disease orillness, delay of the onset of the disease, disorder, or illness, and/orchange in clinical parameters of the condition, disorder, disease orillness, etc., as would be well known in the art.

As used herein, the term “monitoring” refers to assessing thetherapeutic efficacy of a treatment for patients with a cancer. As usedherein, the term “surveillance” refers to the detection of avirus-associated malignancy in subjects, who may or may not have aclinically diagnosed or symptomatic cancer.

As used herein, a subject has an “increased likelihood” of some clinicalfeature or outcome (e.g., recurrence or progression) if the probabilityof the subject having the feature or outcome exceeds some referenceprobability or value. The reference probability may be the probabilityof the feature or outcome across the general relevant subject or patientpopulation. For example, if the probability of recurrence in the generaloropharyngeal cancer population is X % and a particular patient has beendetermined by the methods to have a probability of recurrence of Y %,and if Y>X, then the patient has an “increased likelihood” ofrecurrence. Alternatively, a threshold or reference value may bedetermined and a particular patient's probability of recurrence may becompared to that threshold or reference.

As used herein, “sample” refers to a biological sample containing atumor nucleic acid marker for HPV. The sample may be tissue, cells, orany fluid taken from the human body, e.g., blood, plasma, urine, saliva,etc. In particular embodiments, the sample is a blood-based sample.Accordingly, the sample may be whole blood or components thereof such asserum or plasma.

Also disclosed herein are methods for detection, treatment, andsurveillance of human papilloma virus-associated malignancies and kitsfor accomplishing the same.

Also disclosed herein are methods for quantifying viral nucleic acids inthe circulatory system that are specifically derived from tumors, andfor distinguishing these tumor-derived viral nucleic acids from othersources of circulating viral nucleic acids.

Disclosed herein are DNA amplification methods for quantifying DNAfragments of a target DNA in a sample by size. This can be used, forexample, to detect tumor-derived viral DNA in blood sample anddistinguish it from larger viral DNA from non-tumor sources. Inparticular, disclosed herein are methods of detecting, monitoring, ortreating a human papilloma virus (HPV)-associated malignancy in asubject that involves detecting a presence or absence of at least onecirculating tumor-derived HPV DNA in a sample from the subject. Kits foraccomplishing the same are also provided.

The disclosed method can be performed within thousands of micro-droplets(also referred to herein as droplets) generated through, for example,emulsification and/or water-in-oil droplet partitioning, such as isdescribed in Hindson et al., Anal Chem. 2011 Nov. 15; 83(22):8604-8610,Pinheiro et al., Anal Chem. 2012 Jan. 17; 84(2):1003-1011, andKanagal-Shamanna, Methods Mol Biol. 2016; 1392:33-42, or any othermethod known to those versed in the art to separate a sample intodiscrete and/or volumetrically defined partitions for analysis of tumorderived versus non-tumor derived DNA present in the partitions/sample.In the case of micro-droplets, the size of the droplets can be micro-,nano-, pico-, or femto-scale.

As disclosed herein, the micro-droplets are generated such that theyeach contain at most a single targeted viral nucleic acid. Inembodiments where only two distinct regions of the viral nucleic acidare detected, a micro-droplet containing the targeted viral nucleic acidwill be either single-positive, i.e., positive for one of the detectionsignals, or double-positive, i.e., positive for both of the detectionsignals. As disclosed herein, the relative numbers of thesingle-positive and double-positive micro-droplets and double-positivedroplets provide quantitative information making it possible toquantitatively determine the relative amounts of tumor-derived andnon-tumor derived viral DNA in the sample.

In embodiments where the PCR is used to detect two physically distinctregions, included is a forward and reverse primer pair corresponding toeach detected region. In some embodiments, the detection may beperformed using digital PCR, droplet digital PCR, emulsion PCR ormicro-droplet PCR according to procedures that would be appreciated byone of skill in the art. As disclosed herein, micro-droplets containingtumor-derived circulating viral nucleic acids have fewer positivelydetected viral nucleic acid regions relative to micro-dropletscontaining non-tumor-derived circulating viral nucleic acids. In thesimplest case where only 2 different regions in the viral nucleic acidare targeted for detection, the disclosed method identifiesmicro-droplets containing non-tumor-derived circulating viral DNA asthose that are positive for both of the detection signals employed forthe different targeted regions (double-positive); by contrast, in thissimplest case, micro-droplets containing tumor-derived circulating viralnucleic acid are positive for only one detection signal but not bothsignals simultaneously.

To take an illustrative example, if the target nucleic acid regions fordetection comprise fragments of ˜70-100 bp, two or more target regionsseparated by 100 bp would never be present on the same viral DNAmolecule if the molecule was derived from circulating tumor DNA. Underthis scenario, the simultaneous detection of both target fragmentswithin the same micro-droplet must be due to co-occupancy within thesame micro-droplet of two distinct target fragment molecules, eachcontaining one of the regions targeted for detection. Thus, if theanalyzed samples contain non tumor-derived viral DNA, there will be ahigher frequency of micro-droplets that test positive for two or moretarget fragments than would be expected based on the frequencies of eachtarget region analyzed individually.

For example, one quantitative measurement of the proportion ofnon-tumor-derived viral DNA fragments in the sample is[fraction(droplets double-positive for both detectionsignals)−fraction(droplets positive only for detection signal1)*fraction(droplets positive only for detection signal 2)]. It followsthat the fraction of tumor-derived viral DNA in the sample is[1−[fraction(droplets double-positive for both detectionsignals)−fraction(droplets positive only for detection signal1)*fraction(droplets positive only for detection signal 2)]]. Thecorresponding quantitative measure of tumor-derived viral DNA would be[#(droplets positive only for detection signal 1)+#(droplets positiveonly for detection signal 2)]*[proportion of tumor-derived viral DNAfragments]=[#(droplets positive only for detection signal 1)+#(dropletspositive only for detection signal 2)][1−fraction(droplets positive forboth detection signals)+fraction(droplets positive only for detectionsignal 1)*fraction(droplets positive only for detection signal 2)].These formulae are provided as illustrative examples of how the rawdetection signal data can be converted into measurements oftumor-derived and non-tumor-derived viral DNA in the sample, with theunderstanding there are other formulae that could be utilized for thesame purpose.

The disclosed methods can also be applied in cases where 3 or moreregions, each with its own detection signal, are detected in eachmicro-droplet or other parallelized micro-reaction chamber. In thiscontext, the mathematical formula for quantification of tumor-derivedand non-tumor derived viral DNA can be generalized on the basis of thereasoning provided above for 2 amplified regions.

While the examples above considered detected viral DNA regions of size˜70-100 bp separated by at least about 50 bp, about 60 bp, about 70 bp,about 80 bp, about 90 bp or about 100 bp, it should be understood thatboth the size of the detected DNA fragments and their distance may bevaried and are not fixed in relation to the disclosed methods.

Returning to the embodiment in which two different regions are targetedfor detection, double-positive micro-droplets may on occasion arise fromthe co-incidence of two smaller fragments of viral nucleic acid within asingle micro-droplet, each fragment containing just a single regiontargeted for detection, as opposed to the desired measurement of onelarger fragment encompassing both of the regions targeted for detection.In some embodiments, this possibility can be ruled out, or the extent towhich it is occurring quantified, by comparing the relative frequenciesof double-positive and single-positive droplets, and confirming that thefrequency of double-positive droplets is approximated by the product ofthe frequencies of the single-positive droplets, and reduces inprevalence by the square of the dilution factor when re-analyzed at alower concentration.

In some embodiments, nucleic acids isolated from blood samples areemulsified into micro-droplets such that the vast majority ofmicro-droplets contain either one or none of the viral nucleic acidsthat are being targeted for detection. Experimental methods fordetermining the correct micro-droplet volume and blood sample dilutionfactors are provided herein. Subsequent to emulsification, nucleic aciddetection methods, which may include PCR-based methods, are employed todetect two or more regions of the targeted viral nucleic acid that arephysically separated from one another. In some embodiments, each viralregion being targeted detection is associated with a unique detectionsignal, for example a unique fluorescent color.

Also disclosed herein are methods of detecting ctHPVDNA in HPV-OPSCCpatients. The methods generally involve detecting the presence oftumor-derived viral DNA using a nucleic acid amplification, such aspolymerase chain reaction (PCR), in an emulsified context in which leasttwo distinct regions are amplified to distinguish between tumor-derivedviral DNA, which is fragmented, and non-tumor-derived intact virions,whose DNA is not fragmented. Also disclosed herein are nucleic acidprobes and nucleic acid primers, as well as kits comprising same, foruse in a said method.

Also disclosed herein are methods for identifying the prognosis ofindividuals with HPV-associated OPSCC that can be successfully treatedby de-intensified chemo-radiotherapy (CRT), methods for identifyingtumor-specific biomarkers that are predictive of HPV-associated OPSCCrelapse or recurrence after treatment, and methods of identifying theprognosis of individuals with HPV-associated OPSCC who are at risk forrelapse or recurrence after treatment.

Subjects suitable to be treated by the disclosed methods include, butare not limited to mammalian subjects. Mammals include, but are notlimited to, canines, felines, bovines, caprines, equines, ovines,porcines, rodents (e.g., rats and mice), lagomorphs, primates, humansand the like, and mammals in utero. Any mammalian subject in need ofbeing treated or desiring treatment is suitable. Human subjects of anygender (for example, male, female or transgender) and at any stage ofdevelopment (i.e., neonate, infant, juvenile, adolescent, adult,elderly) may be treated. Subjects may be of any race or ethnicity,including, but not limited to, Caucasian, African-American, African,Asian, Hispanic, Indian, etc., and combinations thereof. It should befurther noted that subject and patient are used interchangeably.

In particular embodiments, the subject has never been diagnosed with orsuffered from a virus-associated malignancy. In other embodiments, thesubject may be diagnosed with, afflicted with, suffering from or at riskfor a virus-associated malignancy. In some embodiments, the subject haspreviously undergone treatment for a virus-associated malignancy. Inother embodiments, the subject may be in remission from avirus-associated malignancy. In some embodiments, the subject is asmoker. In some embodiments, the subject is a non-smoker.

Detection of ctHPVDNA

Methods for determining the level of biomarker nucleic acid, forexample, a ctHPVDNA, such as ctHPV16DNA, in a sample may involve theprocess of nucleic acid amplification, e.g., by PCR, ligase chainreaction (LCR), transcription-based amplification systems, (TAS)self-sustained sequence replication (3SR), nucleic acid sequence-basedamplification (NASBA), strand displacement amplification (SDA) andbranched DNA (bDNA) amplification, Q-Beta replicase, rolling circlereplication, rolling circle amplification, or any other nucleic acidamplification method, followed by detection of the amplified moleculesusing any technique as would be appreciated by one of skill in the art.

In some embodiments, the nucleic acid detection method may be carriedout in micro-droplets or other micro-reaction chamber so that thedetection method can be run in a highly parallelized manner. In someembodiments, micro-droplets or micro-reaction chambers may containeither 0 or 1 copies of the viral DNA region targeted for detection.

In embodiments involving emulsion PCR, a target nucleic acid orpolynucleotide sequence is typically dispersed into micro-droplets. Insome embodiments, it is essential that the target nucleic acids beemulsified at a concentration where each micro-droplet (or droplet)contains either one or zero copies of the target molecule. In the caseof plasma or serum DNA isolated from patient blood samples, theappropriate dilution level can be recognized by assessing the abundanceof a control genomic region and assuring that the frequency of positivemicro-droplets remain less than 10% (<10%). The control genomic regiondetects human (i.e., non-viral) DNA and serves as a quality control forthe sample (since many negative samples will not have any positivesignal in the assay), and will also be used to establish that theconcentration of DNA fragments is appropriate (not to low and not toohigh).

Within each micro-droplet, the principles of conventional PCR alsoapply, where the target molecule is amplified by reaction with at leastone oligonucleotide primer or pair of oligonucleotide primers. Theprimer(s) hybridize to a complementary region of the target nucleic acidand a DNA polymerase extends the primer(s) to amplify the targetsequence. Under conditions sufficient to provide polymerase-basednucleic acid amplification products, a nucleic acid fragment of one sizedominates the reaction products (the target polynucleotide sequencewhich is the amplification product). The amplification cycle is repeatedto increase the concentration of the single target nucleic acid orpolynucleotide sequence within each micro-droplet. The reaction can beperformed in any thermocycler commonly used for PCR.

Methods for setting up a PCR reaction are well known to those skilled inthe art. Any known DNA polymerase, nucleoside triphosphate, buffers,additives/reaction enhancers and conditions for amplification (cycles ofdenaturation, annealing and polymerization) as would be appreciated byone of skill in the art may be used in the PCR reaction.

In some embodiments, the reaction includes a sequence-specific,hydrolysis probe that is conjugated to both a fluorescent molecule and afluorescence-quenching molecule to the reaction mixture to enable thedetection of successful amplification of the target molecule within eachdroplet. The chemical composition of specific probes may vary, butfollow an established method for detecting synthesis-based nucleic acidamplification by those skilled in the art.

The preparation of an emulsion of the PCR reaction can be achieved in avariety of ways that are appreciated by those versed in the art. Oneeffective methodology utilizes a fabricated microfluidic chip that mixesthe aqueous PCR reaction with a lipid solution at controlled pressure togenerate micro-droplets of uniform size. The disclosed method isapplicable to any method for achieving a partitioned PCR reactionmixture that allows the simultaneous detection of two or more viralnucleic acid target molecules.

Following preparation of a PCR reaction mixture that has beenappropriately emulsified or partitioned into droplets, the reactionmixture is subjected to primer extension reaction conditions(“conditions sufficient to provide polymerase-based nucleic acidamplification products”), i.e., conditions that permit for polymerasemediated primer extension by addition of nucleotides to the end of theprimer molecule using the template strand as a template. Cycles ofdenaturation, annealing and polymerization may be performed according toany conditions (e.g., number of cycles, temperatures and duration intime) that would be appreciated by one of skill in the art.

Cycles of denaturation, annealing and polymerization may be performedusing an automated device, typically known as a thermal cycler. Thermalcyclers that may be employed are described elsewhere herein as well asin U.S. Pat. Nos. 5,612,473; 5,602,756; 5,538,871; and 5,475,610.

In other embodiments, non-PCR based applications may be used to detect atarget nucleic acid sequence, for example, where such target may beimmobilized on a solid support. Methods of immobilizing a nucleic acidsequence on a solid support are known in the art and are described inAusubel et al. Current Protocols in Molecular Biology, John Wiley andSons, Inc. and in protocols provided by the manufacturers, e.g. formembranes: Pall Corporation, Schleicher & Schuell, for magnetic beads:Dynal, for culture plates: Costar, Nalgenunc, and for other supports.

Other nucleic acid amplification procedures will be appreciated by oneof skill in the art, such as, but not limited to, LCR, TAS, 3SR, NASBA,SDA, bDNA, and isothermal amplification. The disclosed method is notlimited to the use of amplification by PCR, but rather includes the useof any nucleic acid amplification methods or any other procedures whichmay be useful in amplification of the sequences for the detection and/orquantification of the presence of or expression of one or more of theparticular nucleic acid sequences described herein.

Variations on the exact amounts of the various reagents and on theconditions for the PCR or other suitable amplification procedure (e.g.,buffer conditions, cycling times, etc.) that lead to similaramplification or detection/quantification results are known to one ofskill in the art and are considered to be equivalents.

Detection of the presence of target molecules in a sample/micro-dropletis not particularly limited, and may be accomplished by any techniqueappreciated by one of skill in the art. In some embodiments, detectionmay include hybridization of the target molecule with a target-specificprobe, such as a nucleic acid probe, linked to a fluorescent marker. Inother embodiments, the marker may be a non-fluorescent marker. Thenature of the marker, fluorescent or non-fluorescent, is notparticularly limited and may be any marker or label as would beappreciated by of skill in the art.

The detection and distinguishing of partitions (droplets) that contain atarget molecule from partitions that contain zero target molecules is acritical step in digital PCR. This can be achieved using a variety ofestablished techniques. In some embodiments, micro-droplets are analyzedindividually using a microfluidic channel and a fluorescence detector.Alternatively, advanced microscopy techniques may be implemented tocount positive and negative droplets. The disclosed method may beembodied with any nucleic acid detection method.

While some methods disclosed herein have been implementing using digitalPCR, the disclosed methods can in principle be utilized with any nucleicacid detection method that is capable of detecting single nucleic acidsand distinguishing the size of the detected fragments. For example, suchmethods may include, hybridization of non-amplified target moleculeswith a fluorescent or a non-fluorescent probe, and the like. In any suchembodiments, the disclosed methods can be applied to distinguishtumor-derived viral nucleic acids in circulation from othernon-malignant sources of circulating viral nucleic acid and intactvirions. Particular aspects of the disclosed methods are thesimultaneous detection in a partitioned reaction of at least twofragments of DNA separated by a defined distance in the viral genome,where the presence of both targets in separate partitions is indicativeof tumor viral nucleic acid in a bodily fluid, such as the blood,wherein an increased frequency of co-occupancy of both fragments in thesame partition is indicative of non-malignant viral nucleic acids.

The present invention is more particularly described in the followingexamples that are intended as illustrative only since numerousmodifications and variations therein will be apparent and understood tothose skilled in the art. Examples of how the application of thisspecific and sensitive method for detecting tumor viral nucleic acids ina bodily fluid, such as blood, can be used to predict patient prognosisduring cancer treatment, and to identify patients who are at the highestrisk of disease recurrence among a cohort of patients who are clinicallyasymptomatic and thought to be in disease remission are provided below.

A number of embodiments of the invention have been described. Thedisclosure is further described in the following examples, which do notlimit the scope of the invention described in the claims.

EXAMPLES Example 1: Digital PCR Assay to Detect HPV viral DNA inCirculating Cell-Free DNA

Provided here is an embodiment of the disclosed methods that usesdigital emulsion PCR to detect tumor-derived viral HPV DNA in thecirculating cell-free DNA isolated from blood. The methodologicaldetails described in this example, for example the nucleic acidamplification and detection methods used, are included solely toestablish that the invention has been reduced to practice. Themethodological details provided in this example related only to thisparticular embodiment are not to be construed as limiting the invention,as described in the claims and summary sections of this document.

Materials

Reagents: Cell-Free DNA BCT tubes, RUO (Streck catalog No. #218962);QIAamp™ Circulating Nucleic Acid Kit, Catalog #55114; dPCR Supermax®Bio-Rad catalog no #186-3024; Eppendorf™ 96-Well twin.tec™ PCR Plates;Fisher scientific catalog No. #E951020362; Pipet tips (Bio-Rad catalogNo. #186-4121 or #186-4120); Cartridge (Bio-Rad catalog No. #186-4109 or#186-4108); Sealing foil (Bio-Rad catalog No. #181-4040); Optional:VacConnectors® (Qiagen Cat No./ID: 19407). This extra connector isuseful in case something goes wrong with connectors available in QIAampCirculating Nucleic Acid Kit, Catalog #55114; Bovine Serum Albumin(BSA); Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific Catalognumber: #Q32851); Qubit Assay Tubes (Thermo Fisher Scientific Catalognumber: #Q32856); Falcone 15 ml Conical Centrifuge Tubes (CorningCatalog No. #352096); Falcon™ 50 ml Conical Centrifuge Tubes (CorningCatalog No. #352098); Disposable sterile 5 ml, 10 ml and 25 mlSerological Pipets (any good brand); Sterile PCR tubes (Any good brand);Sterile filter tips for capacity 2 μl, 10 μl, 200 μl and 1200 μl (anygood brand); primers and probes.

Instruments: Microcentrifuge (suitable for 1.5 ml Eppendorf tubes, e.g.Eppendorf 5424 Microcentrifuge); Centrifuge (Suitable for 15 ml falcontubes, e.g. Eppendorf Centrifuge 5810 R); Qubit Fluorimeter (ThermoFisher Scientific Catalog No. #Q33226); Deep 96 well thermocycler(Bio-Rad's C1000 Touch™ Thermal Cycler with 96-Deep Well Reaction Module#1851197); Heating block for drying 1.5 ml Eppendorf tubes (DenvilleScientific catalog No. #10540); Water bath (should have sufficient spacefor incubating twenty-four 50 ml Conical Centrifuge Tubes as 24 samplescan be processed at one time); Automated droplet generator (Bio-Radcatalog No. #186-4101); Bio-Rad QX200™ Droplet Reader Catalog No.#1864003; Portable Pipet-Aide XP Pipette Controller (DrummondScientific, Catalog No. 4-000-101); Pipette (capacity: 2 μl, 10 μl, 20μl, 200 μl and 1000 μl; any good brand); 8-channel pipette (any goodbrand, e.g. Eppendorf Catalog No. #3125000010).

Methods

Blood Collection: Blood is collected in Cell-Free DNA BCT tubes, RUO(Streck catalog No. #218962).

Plasma Extraction: Collected blood from step I should ideally beprocessed on the same day to extract plasma. Same tube can becentrifuged at 2000×g for 10 min at room temperature (RT). Supernatantis transferred to new Falcon™ 15 mL Conical Centrifuge Tubes. Careshould be taken to avoid taking middle whitish layer below plasma.Centrifuge the tube again for 10 min at 2000×g at RT. Supernatant isthen transferred to a new Falcon™ 15 ml Conical Centrifuge Tubes. Theplasma is at −80° C. freezer till further use. NOTE: sometimes 10 min.centrifuge does not lead to clear separation of plasma layer. In suchsituation, sample should be centrifuged for another 10 min before takingout the plasma. Or, alternatively centrifugation at first step can bedone for 15-20 min. Record the sample if plasma looks red. Sometimesextra processing of the sample is required during PCR step because ofthe hemolysis. Blood should be discarded in 10% bleach or autoclaved oraccording to any other institution/company approved protocol.

Plasma cell free DNA (cfDNA) Extraction: The stored plasma samples arethawed at 37° C. in a water bath for about 5 min. A Qiagen kit (QIAampCirculating Nucleic Acid Kit, Catalog #55114) is used to extract DNAusing manufacturer's protocol with the following modifications: Standardvacuum available in laboratory can be used in the protocol; The cfDNA iseluted in 2 steps—first with 100 μl elution buffer and then 75 μlelution buffer if plasma volume is more than 3 ml. Elution can be donein lower volume if the collected plasma volume is less to avoidexcessive dilution of the cfDNA; and it was observed that incubation ofthe column for 3 min. after adding elution buffer (as suggested in theprotocol) does not lead to the complete extraction of cfDNA. Generally,a 30 min to 1 h incubation at RT generally follows both elution steps.The cfDNA is quantified on a Qubit fluorimeter. (Note: Generally, 2 μlof the eluate is sufficient to be used for quantification purpose). Theeluted cfDNA is stored at −20° C. freezer until further use. Note: ThecfDNA recovered at first elution is used for all experiments andcalculations. The cfDNA recovered in the second elution step is usedonly if cfDNA at first elution is exhausted. If a more concentratedcfDNA is required for any purpose like NGS, then the sample can beconcentrated using speed vacuum.

dPCR: The dPCR involve three steps—Droplet generation; PCR; and dropletreading.

Droplet Generation: Prepare 25 μl reaction for each sample customized asper the following composition (Reagents: dPCR Supermax® Bio-Rad catalogno #186-3024; any nuclease free PCR grade water can be used; otherreagents like forward primer, reverse primer and probe are designed byuser)

TABLE 1 Reaction Mixtures Working Final 1x 1x No template Componentstock Conc. (sample) control (NTC) Primers Mixture 22.5 μM  0.9 μM   1μl   1 μl (4) each each Probes Mixture 6.25 μM 0.125 μM  0.5 μl  0.5 μl(2) each each Albumin 10% 0.4%   1 μl   1 μl Betaine   5M  0.4 μM   2 μl  2 μl Trehalose 1.25M 0.16M  3.2 μl  3.2 μl dPCR Supermix 2x 1x 12.5 μl12.5 μl DNA sample 1x/Diluted N/A  4.3 μl — PCR grade Adjustable N/A — 4.3 μl Water Total   25 μl   25 μl

The primer and probe sequences for the ctHPVDNA assay described here areas follows:

TABLE 2 Primers and Probes Variant Primers/Probes HPV16283_HPV-16_F1 For: TGACTCTACGCTTCGGTTG (SEQ ID NO: 1) Fragment 1284_HPV-16_F1 Rev: GCCCATTAACAGGTCTTCC (SEQ ID NO: 2)420_HPV-16_F1 Probe_FAM-ZEN_v2:CGTACAAAdCACACAGTAGACATTCGTAC (SEQ ID NO: 3) HPV16424_HPV16_F2_For: GGTTTGTAACATCCCAGGC (SEQ ID NO: 4) Fragment 2425_HPV16_F2_Rev: GTGTATTTTTTAAGGGGATCTTCTT (SEQ ID NO: 5)421_HPV16_F2_HEX_LNA: CACCT(+C)(+C)A(+G)CACC (SEQ ID NO: 6)″(+N)″ denotes LNA™ base

The Primers mixture working stock is assembled by combining 22.5 μL eachof the four primers (100 μM concentration) into a tube and adding 10 μLnuclease-free water to achieve a final concentration of 22.5 μM for eachprimer.

The Probe mixture working stock is assembled by combining 6.25 μL ofeach probe (100 μM concentration) into a tube and adding 87.5 μLnuclease-free water to achieve a final concentration of 6.25 μL for eachprobe.

About 22-23 μl of the above reaction mixture is loaded on a 96 wellplate (Eppendorf™ 96-Well twin.tec™ PCR Plates, Fisher scientificcatalog No. #E951020362) using Multichannel Pipetors (Note: Instrumentuses only 20 μl from each well. An extra volume is added to avoid anypipetting error) Droplets are generated using automated dropletgenerator (Bio-Rad catalog No. #186-4101) following manufacturer'sprotocol. Reagents required at this step are pipet tips (Bio-Rad catalogNo. #186-4121 or #186-4120) and cartridges (Bio-Rad catalog No #186-4109or #186-4108). The sample plate is sealed with sealing foil (Bio-Radcatalog No. #181-4040) following manufacturer's protocol. Note: Thevolume of cfDNA+water should be 4.3 μl. Generally, there is no issue byusing 4.3 μl of cfDNA sample in the reaction but sometimes the allelecopy number is too high and it results in streaking of the positivedroplet across the axis and many dual positive droplets. In such cases,the run should be repeated by using less sample and remaining volume canbe adjusted with water. Dilution gives better quantification of allelefrequency in such cases. A linear relationship across 5 orders ofmagnitude has been observed in HPV16 copy number ranging from 5 copiesto 50,000 copies per 20 μl reaction. Care should be taken to seal theplate properly. If the plate is not sealed properly then it will lead tosample evaporation during PCR step.

PCR: The PCR thermocycling was performed using the protocol as describedbelow.

TABLE 3 PCR conditions Temperature, Ramp Number of Cycling step ° C.Time Rate Cycles Enzyme activation 95 10 sec 2° C./sec 1 Denaturation 9430 sec 40 Annealing 60 1 min 40 Extension 68 1 min 40 Enzyme 98 10 min 1deactivation Hold (optional) 4 Infinite 1 Use a heated lid set to 105°C. and set the sample volume to 40 μl

All wells should be observed visually after PCR and before reading theplate on droplet reader. The copy numbers observed during dropletreading may not be real if there is any well where sample is evaporatedbecause of improper sealing. It is good to leave the plate in thethermocycler for about 15-20 min after PCR is done. It allows thetemperature of the plate to come down at more controlled way and itavoids droplet malformation. Sometimes malformation of the dropletsbecause of the static current can be avoided by touching hand with othermetallic surface before taking out the plate from thermocycler. Itshould be made a routine practice for better reproducibility of theresults. The PCR plate can be stored overnight after PCR and reading canbe done next day morning if time is limited. However, finishingeverything in one day is best practice.

Droplet Reading: The droplet reader (Bio-Rad QX200™ Droplet ReaderCatalog No. #1864003) is used to read signal in droplets following themanufacturer's protocol. Note: Discarding Oil waste: The composition ofdroplet reader oil is proprietary of Bio-Rad. A typical waste profilecontains fluorinated oils (95%), water (5%), bleach (<0.5%), proteins,nucleic acids, and fluorescent dye (<0.1%). A proper disposal should beplanned accordingly.

Data Analysis: The copy number calculations should be done following themanufacturer's guidelines. An example of the dPCR ctHPVDNA assay readoutis shown in FIG. 1A. The following parameters were set for calculationof FAM-single positive, HEX-single positive, and FAM+HEX double positivedroplets. In the FAM channel, a cutoff of 700 is used to separate thenegative from positive droplets. Similarly in the HEX channel, a cutoffof 3000 is used to separate the negative from positive droplets. TheFAM+HEX double positive droplets are those with >700 fluorescenceintensity in the FAM channel and >3000 fluorescence intensity in the HEXchannel. The double negative droplets have FAM fluorescence <700 and HEXfluorescence <3000.

Poisson statistics is used to calculate the copies of each fragment inthe reaction individually, using the following formula:

#copies=#total droplets*ln(#total droplets/#signal negative droplets).

This is calculated first for fragment 1 (FAM positive) and for fragment2 (HEX positive).

Extensive control assays have been run to determine the level ofexperimental noise. Based on these controls, the following criteria wereused to determine the number of copies of the target fragment(s) in theassay reaction.

TABLE 4 Control Criteria HPV16 copies/ 20 μl reaction Considered as 0Zero Below 3 False positive and should be considered as zero 3-5 Assayshould be repeated to confirm the value Above 5 Considered as positivefor HPV16 Any positive value in Assay should be follow up patient whenrepeated to confirm previous HPV16 the value values are negative

These copy number values can be used to calculate the frequency of adroplet possessing the target fragment: Pr (frag)=(#positivedroplets/#total droplets). The frequency of double-positive dropletsthat are expected if the sample consists of fragmented, circulatingtumor viral DNA is estimated as: Pr (double positive)=Pr (frag1)*Pr(frag2). In contrast, if the Pr (dual positive)>2*Pr (frag1)*Pf (frag2),then the sample was considered to contain non-fragmented viral DNA thatis not tumor-derived. If Pr (double positive)>Pr (frag1) or Pr (doublepositive)>Pr (frag2) it was interpret that the sample is negative forcirculating tumor-derived viral nucleic acids. If Pr (frag1) AND Pr(frag2)>2*Pr (double positive), then the sample was considered positivefor circulating tumor-derived viral nucleic acids, although there isevidence for coexistent non-tumor derived viral nucleic acids. Raw datafor this assay applied to experimental controls and plasma DNA from acohort of 12 patients with HPV+ oropharyngeal cancer is shown in FIG.1A-1B.

For the HPV16 F1 assay, cutoff of 700 was set on y-axis (FAM channel).Any droplets above 700 are considered as positive droplets for HPV16.For the HPV16 F2 assay, a cutoff of 3000 was set on x-axis (Hexchannel). Any droplet above 3000 was considered as positive droplet forHPV16 F2. Occasionally, the patterns of the dPCR readout look unusual.This can be because of a bad sample (unanalyzable cfDNA) or a bad dPCRrun. Such sample should be repeated to fix the problem. Data from suchsamples cannot be used for interpretation. If sample quality is notimproved with any available strategies then sample should be categorizedas unanalyzable DNA or bad sample. Some strategies to improve the samplequality are as below. The presence of Heparin in the blood sample caninterfere with the dPCR reaction. Treating the sample with BacteroidesHeparinase I (New England Biolabs cat no. #P0735S) using manufacturer'sprotocol improves the quality of sample. Excessive hemolysis sometimesinterferes with the dPCR. An improvement has been observed in the assayreadout by adding 0.4% bovine serum albumin Some of the representativedot plots from bad samples are provided as separate file with suggestedsolutions.

Using an endogenous genomic sequence as a positive control for the dPCRreaction is essential for validating sample quality. dPCR-baseddetection of a target sequence in the ESR1 gene was utilized as apositive control for sample quality. The assay is described below:

TABLE 5 Variant Primers and Probes Genomic132_CtrIESR1_F2: ATCTGTACAGCATGAAGTGCAAGA (SEQ ID Control NO: 7)(ESR1 locus) 133_CtrIESR1_R2: CTAGTGGGCGCATGTAGGC (SEQ ID NO: 8)094_CtrIESR1_LNA2-TET_probe_WT:T(+C)(+T(+A)T(+G)(+A)(+C)CTG (SEQ ID NO: 9) ″(+N)″ denotes LNA™ base

TABLE 6 Setting PCR Reaction Component Working stock Final conc. 1x(sample) 1x (NTC) Forward primer 22.5 μM  0.9 μM   1 μl   1 μl Reverseprimer 22.5 μM  0.9 μM   1 μl   1 μl Probe 6.25 μM 0.25 μM   1 μl   1 μlBSA 10% 0.4%   1 μl   1 μl dPCR Supermix 2x 1x 12.5 μl 12.5 μl DNAsample Neat/diluted N/A  8.5 μl — PCR grade Water Adjustable N/A —  8.5μl Total   25 μl   25 μl

TABLE 7 PCR Conditions Temp, Ramp Number Cycling step ° C. Time Rate ofCycles Enzyme activation 95 10 sec 2° C./sec 1 Denaturation 94 30 sec 40Annealing/Extension 60 1 min 40 Enzyme deactivation 98 10 min 1 Hold(optional) 4 Infinite 1 Use a heated lid set to 105° C. and set thesample volume to 40 μl

Work Flow for the analysis of plasma DNA samples:

1) Test samples using ESR1 genomic control dPCR assay, to evaluateamplifiable DNA and sample concentration.

2) Test the samples for the dual fragment HPV16 assay (HPV16ZENv2)

4) If sample is negative for HPV16 then perform the HPV multiplexedassay (HPVmultiplexed_v1)

5) Perform the duplex assay to know specific HPV variant

Example 2: Multiplexed dPCR Assay To Detect 5 Distinct HPV Sub-Strains(HPVmultiplexed_v1)

The disclosed methods were applied to sub-strains of a given virus.While the embodiment here relates to sub-strains of the HPV virus, themethod can be readily applied to other viral sub-strains usingestablished techniques known to those versed in the field. Variantsincluded in the described embodiment of the method are HPV16, HPV18,HPV31, HPV33 and HPV35

HPV16 probe was tagged with FAM-Zen while others with HEX (LNA version).

TABLE 8 Primers and Probes Variant Primers and Probes HPV16283_HPV-16_For: TGACTCTACGCTTCGGTTG (SEQ ID NO: 1)284_HPV-16_Rev: GCCCATTAACAGGTCTTCC (SEQ ID NO: 2)420_HPV-16_Probe_FAM-ZEN_v2:CGTACAAAGCACACACGTAGACATTCGTAC (SEQ ID NO : 3) HPV18401_HPV18_For: TGAAGCCAGAATTGAGCTAG (SEQ ID NO: 10) 422_HPV18_Rev_v2:AGGACAGGGTGTTCAGAA (SEQ ID NO: 11)403_HPV18_LNA_HEX-Probe: CA(+G)A(+C)(+G)AC(+C)TTCG (SEQ ID NO: 12) HPV31404_HPV31_For: AGCACACAAGTAGATATTCGC (SEQ ID NO: 13)405_HPV31_Rev: TAGTAGAACAGTTGGGGCA (SEQ ID NO: 14)406_HPV31_LNA_HEX-Probe: TAA(+C)(+A)G(+C)T(+C)(+T)TG(+C) (SEQ ID NO: 15)HPV33 407_HPV33_For: TAACACCACAGTTCGTTTATGT (SEQ ID NO: 16)423_HPV33_Rev_v2: ACAATATTCACTGTGCCCATA (SEQ ID NO: 17)418_HPV33_LNA_HEX-Probe_v2:TG(+A)C(+C)(+T)A(+C)G(+A)(+A)CC (SEQ ID NO: 18) HPV35410_HPV35_For: TGAGGCGACACTACGTC (SEQ ID NO: 19)411_HPV35_Rev: GTGCCCATTAATAAATCTTCCAA (SEQ ID NO: 20)419_HPV35_LNA_HEX-Probe_v2: AG(+A)G(+C)(+A)CA(+C)(+A)CAT (SEQ ID NO: 21)″(+N)″ denotes LNA™ base

Reaction Mixture

Primer mixture: Mix equal volume of 10 primers each at 100 μM (eachprimer diluted 1:10 in the mixture). Probe mixture: Mix 6.25 μl of eachof five probes in a tube and add 68.75 μl water (final concentrationwill be 6.25 μM each in 100 μl volume)

TABLE 9 Reaction Mixtures Working 1x 1x Component stock Final Conc.(sample) (NTC) Primers Mixture   10 μM each  0.9 μM each 2.25 μl 2.25 μlProbes Mixture 6.25 μM each 0.125 μM each  0.5 μl  0.5 μl Albumin 10%0.4%   1 μl   1 μl Betaine   5M  0.4 μM   2 μl   2 μl Trehalose 1.25M0.16M  3.2 μl  3.2 μl dPCR Supermix 2x 1x 12.5 μl 12.5 μl DNA sampleNeat/Diluted N/A 3.55 μl — PCR grade Water Adjustable N/A — 3.55 μlTotal   25 μl   25 μl

TABLE 10 PCR conditions Temperature, Ramp Number of Cycling step ° C.Time Rate Cycles Enzyme activation 95 10 sec 2° C./sec 1 Dentaturation94 30 sec 40 Annealing 60 1 min 40 Extension 68 1 min 40 Enzyme 98 10min 1 deactivation Hold (optional) 4 Infinite 1 Use a heated lid set to105° C. and set the sample volume to 40 μl

Note: An LNA™-FAM probe was also designed to amplify common region inall known HPV16 variants with same primers (No. 283 & 285). This was notused in the multiplexed assay.

(SEQ ID NO: 22) 413_HPV16_LNA_FAM-CommonProbe:CA(+C)A(+C)(+G)(+T)A(+G)(+A)CAT

Example 3: Duplex dPCR Assay to Detect Specific HPV Variants (HPV18&33_v1 and HPV 31&35_v1)

This assay was designed to know the identity of specific HPV variants inpatient samples.

TABLE 11 Primers and Probes Variant  Primers Probe Set 1 HPV18401_HPV18_For: TGAAGCCAGAATTGAGCTAG & (SEQ ID NO: 10) HPV33422_HPV18_Rev_v2: AGGACAGGGTGTTCAGAA (SEQ ID NO: 11)407_HPV33_For: TAACACCACAGTTCGTTTATGT (SEQ ID NO: 16) 423_HPV33_Rev_v2:ACAATATTCACTGTGCCCATA (SEQ ID NO: 17)403_HPV18_LNA_HEX-Probe: CA(+G)A(+C)(+G)AC + CTTCG (SEQ ID NO: 12)426_HPV33_LNA_FAM-Probe: TG(+A)C(+C)(+T)A(+C)G(+A)(+A)CC (SEQ ID NO: 18)Set 2 HPV31 404_HPV31_For: AGCACACAAGTAGATATTCGC (SEQ ID & NO: 13) HPV35405_HPV31_Rev: TAGTAGAACAGTTGGGGCA (SEQ ID NO: 14)410_HPV35_For: TGAGGCGACACTACGTC (SEQ ID NO: 19)411_HPV35_Rev: GTGCCCATTAATAAATCTTCCAA (SEQ ID NO: 20)406_HPV31_LNA_HEX-Probe: TAA(+C)(+A)G(+C)T(+C)(+T)TG(+C) (SEQ ID NO: 15)427_HPV35_LNA_FAM-Probe: AG(+A)G(+)(+A(CA(+C)(+A)CAT (SEQ ID NO: 21)″(+N)″denotes LNA™ base

Reaction Mixture

Primer mixture: Mix 22.5 μl of each of the four primers and add 10 μlwater (22.5 μM each primer in the mixture). Probe mixture: Mix 6.25 μlof both probes in a tube and add 87.5 μl water (final concentration willbe 6.25 μM each in 100 μl volume).

TABLE 12 Reaction Conditions Working 1x 1x Component stock Final Conc.(sample) (NTC) Primers Mixture 22.5 μM each  0.9 μM each   1 μl   1 μlProbes Mixture 6.25 μM each 0.125 μM each  0.5 μl  0.5 μl Albumin 10%0.4%   1 μl   1 μl Betaine   5M  0.4 μM   2 μl   2 μl Trehalose 1.25M0.16M  3.2 μl  3.2 μl dPCR Supermix 2x 1x 12.5 μl 12.5 μl DNA sampleNeat/Diluted N/A  4.3 μl — PCR grade Water Adjustable N/A —  4.3 μlTotal   25 μl   25 μl

TABLE 13 PCR conditions Temperature, Ramp Number of Cycling step ° C.Time Rate Cycles Enzyme activation 95 10 sec 2° C./sec 1 Dentaturation94 30 sec 40 Annealing 60 1 min 40 Extension 68 1 min 40 Enzyme 98 10min 1 deactivation Hold (optional) 4 Infinite 1 Use a heated lid set to105° C. and set the sample volume to 40 μl

Example 4: Application to Patient Blood Samples in Prospective ClinicalTrials

Blood samples were analyzed from healthy volunteers, non-virusassociated cancer, and HPV+ cancer. Circulating tumor HPV DNA copynumbers, as measured using the assay technology described in Example 1,are shown in FIG. 2. These data indicate that the ctHPVDNA blood testhas 100% specificity and 93% sensitivity for identifying patients withHPV+ cancer.

Blood samples were analyzed from patients enrolled in two prospectivephase II clinical trials. The disclosed method for specificallydetecting tumor-derived viral HPV DNA in the blood was integrated intothe design of both of these clinical trials. The findings from theselongitudinal clinical studies illustrate the applicability and utilityof the disclosed method both for personalizing patient therapy and forcancer surveillance.

Summary of patient cohort and clinical trial design. Two prospectivephase II clinical trials were completed that evaluated the efficacy of ade-intensified chemo-radiation therapy regimen in low risk OPSCC. InLCCC 1120 (NCT01530997) 45 patients were treated with de-intensifiedCRT. Eligible patients had HPV-positive and/or p16-positive OPSCC,T0-T3, N0-N2c, MO, and <10 pack years of smoking. Patients received 60Gy of Intensity Modulated Radiotherapy (IMRT) with concurrent weeklyintravenous cisplatin (30 mg/m²). All patients had biopsy of the primarysite and underwent a selective neck dissection to encompass nodallevel(s) that were positive pre-treatment, within 4 to 14 weeks afterCRT. The primary endpoint of LCCC 1120 was the rate of pCR, which was86% (37/43) and the 3-year cancer control and overall survival was 100%and 95%, respectively. In addition to excellent cancer control, patienthad less toxicity and reported excellent quality of life and lowersymptom burden as compared to standard of care CRT (70Gy). In asecond-generation phase 2 study (LCCC 1413, NCT02281955) a 12-weekpost-treatment PET/CT was used to guide the use of biopsy/neckdissection (i.e. post CRT surgical evaluation was not mandatory, butguided by imaging). Of the 113 patients enrolled, 82 patients have had aminimum of 1-year follow-up and the 2-year actuarial cancer control andoverall survival in these 82 patients is 90% and 95% respectively (againexcellent results). Patients continue to report good recovery of qualityof life at 1 year, and thus supports the concept that dose de-escalationcan improve the therapeutic ratio. A third generation phase 2 study(LCCC 1612, NCT03077243) is currently being conducted, and has enrolled53 of an expected 120 patients. Blood samples from 113 patients wereprospectively analyzed to detect tumor-derived plasma viral HPV DNA.

Levels of tumor-derived viral HPV DNA in the blood, as quantified by thedisclosed method, correlate significantly with HPV viral DNA in primarytumors. In 63 patients with HPV-OPSCC, serial levels of tumor-derivedviral HPV DNA (pre- and weekly during-RT) were prospectively quantifiedin patient blood samples using the disclosed method (FIG. 3A). 49patients had detectable tumor-derived viral HPV DNA in the circulationprior to treatment. In all 49 patients, the levels of circulating HPVDNA had dropped significantly by week 6 (FIG. 3B) following theinitiating of chemo-radiation therapy, diminishing to undetectablelevels in a majority (90%, n=44/49) of patients by the end of thetherapeutic regimen. Across patients, the peak levels of tumor-derivedviral HPV DNA in the blood, ranged from 10 copies/mL to ˜30,000copies/mL. Moreover, distinct clearance kinetics of tumor-derived viralHPV DNA was observed in the blood during CRT treatment. In one groupthere was rapid clearance kinetics (FIG. 3C), with >95% of the peaktumor-derived viral HPV DNA in the blood being cleared by day 28 oftherapy. In a second group there was delayed clearance of tumor viralHPV DNA in the blood (FIG. 3D), which may be associated with poor ordelayed response to therapy.

The disclosed method was further validated by correlating levels oftumor-derived viral HPV DNA in the blood with analysis of matchedprimary tumors in a cohort of 20 patients (FIG. 4). Normalized HPV DNAcopies in tumor biopsy material correlates strongly with HPV DNA copiesas measured by next-generation sequencing (FIG. 4A). Moreover, thequantity of tumor-derived viral HPV DNA in blood correlatessignificantly with HPV DNA copy number in the matched tumor biopsy,after normalizing to the overall tumor burden in the patient (FIG. 4B).Using next-generation sequencing, cancers with integration of HPV intothe cancer cell genome were identified (FIG. 4C-4D). Higher copy numberof HPV DNA in tumor biopsy material correlated with a high likelihood ofepisomal (non-integrated) HPV DNA in the matched primary tumor (FIG.4E). Similarly, higher copy numbers of tumor-derived viral HPV DNA inthe blood correlated with a higher likelihood of episomal(non-integrated) HPV DNA in the matched primary tumor (FIG. 4F). Thesefindings validate the disclosed methods by establishing that thequantification of viral DNA in the blood using the disclosed methodscorrelates significantly with measurements of viral DNA in matched tumortissue. These observations also demonstrate the disclosed method canmonitor the presence and clearance of tumor-derived HPV viral DNA inlongitudinal analysis of patient blood samples.

Predict clinical risk in cancer patients. The disclosed method wasapplied to monitor tumor-derived viral HPV DNA in the blood before andduring therapy in 63 patients enrolled in the clinical trial describedabove. A Favorable Profile was defined as having abundant ctHPV16DNApeak levels of tumor-derived viral HPV DNA in the blood (>200 copies/mL)and rapid clearance kinetics (≤2% of the peak value by week 4) (FIG.5A). 18 out of 63 evaluable patients (29%) had a Favorable Profile (FIG.5B), and none of these 18 patients have recurred (regardless of smokingstatus) (FIG. 6A-B). An Unfavorable Profile was defined as (i)undetectable pre-treatment tumor-derived viral HPV DNA in the blood,(ii) low peak values of tumor-derived viral HPV DNA in the blood (≤200copies/mL), or (iii) >2% of the peak value by week 4 (40Gy) (FIG. 5A-B).Remarkably, patients with a Favorable Profile exhibited 100% diseasecontrol in non-smokers (60Gy) and heavy smokers (60-70Gy). In contrast,heavy smokers (>10 pack year) with an Unfavorable Profile had a verypoor regional disease control rate of 45% at 12 months after completingtherapy (FIG. 6A-B). These observations demonstrate the clinical utilityof applying the disclosed method to quantify tumor-derived viral DNA inthe blood as a biomarker to predict clinical risk among virus-associatedcancer patients. Moreover, these findings indicate that a real-timeassessment of circulating tumor-derived viral DNA can be utilized topersonalize the intensity of therapy for patients based on theirinferred risk.

Early detection of HPV-positive cancer recurrence in healthy patientsthat are asymptomatic and considered disease-free using existingclinical procedures. The typical surveillance schedule afterchemo-radiation therapy is clinical examinations (physical examinationand fiber optic nasopharyngolaryngoscopy) every 2 to 6 months for thefirst 5 years. Most head and neck oncologist also obtain PET/CT every 6to 12 months. There are currently no available surveillance blood testsfor patients with HPV-associated OPSCC. The availability of highlysensitive blood-based surveillance test would aide in early detection ofcancer recurrence (prior to clinical or radiographic findings) andimprove the value of cancer surveillance in this population by reducingthe frequency of office visits and the use of expensive radiologicalimaging studies. To date 73 patients have been prospectively surveilledwith HPV-associated OPSCC (FIG. 7). Blood samples were obtained witheach follow-up visit regardless of clinical findings. Plasma ctHPV16DNAhas become detectable in follow up after treatment in 13 patients(median copies/ml and range) of which 9 have had clinical/radiographicevidence of cancer recurrence. These patients with detectable ctHPV16DNAafter treatment were asymptomatic and had radiographic examinationsconfirming very early, low volume cancer recurrences. An illustrativecase example is presented in FIG. 8. Here, the patient had alreadycompleted curative-intent therapy and was completely asymptomatic withno evidence of disease. However, he developed a positive result fortumor-derived viral HPV DNA in the blood in June 2017. Soon thereafter,he was examined by an oncologist and determined to have no evidence ofdisease. Three months later, he had another follow up visit and theoncologist did not identify any evidence for disease recurrence onphysical exam. However, the patient reported some neck/shoulder pain,which was believed to be musculoskeletal in nature. A neck/shoulder MRIwas ordered, and on this exam—4 months after the initially positiveblood test—an isolated abnormally enlarged lymph node was identified inthe neck. This abnormal mass was subsequently biopsied and proven to berecurrent HPV+ oropharyngeal cancer.

There are 4 patients in the study who have detectable HPV DNA in theblood using the disclosed methods, yet have no clinical/radiographicevidence of disease and are being closely followed for recurrence. Nocancer recurrences have been detected in patients with undetectablectHPV16DNA. In summary the negative predictive value and positivepredictive value of the plasma ctHPV16DNA assay in detecting cancerrecurrence is 100% and 70%, respectively. These observations suggestthat plasma ctHPVDNA is exquisitely specific and sensitive for the earlydetection of HPV-associated cancer. Application of the disclosed methodas part of clinical care may improve the effectiveness and reducing thecost of cancer surveillance for patients with HPV-associatedoropharyngeal cancer.

Example 5: Plasma Circulating Tumor HPV16DNA as a Biomarker forTreatment of HPV-Associated OPSCC

The RTOG 0129 study demonstrated that exposures influence clinical riskin oropharyngeal cancer. HPV-positive patients tend to do well, andHPV-negative patients with extensive smoking history do poorly. Moststudies, including RTOG 0129, also demonstrate an intermediate prognosisfor HPV+ cancers that develop in heavy smokers. This is shown in FIG. 8.

Genetic biomarkers can improve clinical risk stratification. Forexample, a subset of tumors in HPV+ non-smokers may be exceptionallysensitive to therapy, whereas, there may be smokers with HPV+ cancersthat have more HPV-like biology, and others that may have moretobacco-like biology. Biomarkers may be able to identify these subgroupsbetter than clinical parameters alone.

Plasma ctHPVDNA is detected in a majority of HPV-OPSCC patients. Assuch, ctHPV DNA can be a biomarker of tumor burden, and as importantly,response kinetics. Plasma circulating tumor HPV DNA can also informdecisions regarding who is appropriate for de-escalated therapy ofHPV-associated OPSSC.

A digital PCR assay has been developed for HPV16 DNA that is: highlyspecific, in that the assay does not cross-detect HPV-18, -31, -33, or35, and has very low background signal; linear over 5 orders ofmagnitude in copy number (5-50,000 copies); precise and has exceptionalreproducibility; and ultra-sensitive and can detect as few as 6 copiesof HPV16 with ˜80% sensitivity. This is shown in FIGS. 1A and 1B. FIG.1A shows an example readout of this assay where positive dropletsindicate the presence of individual HPV16 DNA molecules, that are wellseparated from the negative droplets in a reaction. FIG. 1B shows the95% confidence interval of a linear regression, demonstrating incrediblelinearity and precision, with assay variability only becoming an issuein the 10 target copy range. This assay can detect as few as 6 targetmolecules of HPV16 DNA using an assay threshold that gives no falsepositives.

A study population including 64 patients with biopsy-proven HPV-positiveOPSCC that overexpressed p16 (IHC) and/or were HPV ISH positive. Allpatients received definitive chemo-radiation (no induction chemo). 54patients (84%) enrolled in a prospective CRT de-intensification trial(60Gy IMRT+weekly Cisplatin 30 mg/m²). Of these, 46 patients wereclinically favorable (<T4 and 10 pack-years tobacco), and 18 wereclinically unfavorable (T4 or >10 pack-years tobacco). No patients hadN3 or M1 disease. Research bloods were collected pre-treatment, weeklyduring CRT, and at post-treatment clinic visits (-450 blood samplesanalyzed).

Plasma ctHPV16DNA levels were measured during chemo-radiotherapy. PlasmactHPV16DNA is responsive to CRT and in most cases is “cleared” aftertreatment. Its potential as a biomarker of treatment efficacy, andcorrelation of peak-ctHPV16DNA levels with smoking status, diseaseburden, and tumor HPV copy number was evaluated. This regimen is shownin FIG. 10, and normalized levels of ctHPV16DNA is depicted in FIG. 3A.

Plasma ctHPV16 DNA was detected in 77% of the patients, and FIG. 11indicates the peak value for each patient. As shown in FIG. 11, broadrange of values was observed, with as few as 10 copies per mL to as highas 30,000 copies per mL being observed. There were also 23% of patientswho did not have any detectable HPV16 DNA in plasma. Furthermore, anysignificant correlation between the amount of HPV16 in plasma andsmoking status was not seen.

However, plasma ctHPV16 levels did not exhibit significant correlationwith disease burden, as shown in FIG. 3C. Many different ways for acorrelation between disease burden and peak HPV16 DNA values were lookedinto, one could not be found. However, by analyzing a 25 patient subsetin whom tumor sequencing and plasma HPV16 DNA had been matched, a modestbut statistically significant correlation between plasma circulatingtumor HPV16 and copy number of HPV16 in the tumor was identified, shownin FIG. 3D. Thus, the number of HPV16 copies in plasma for a particularpatient may reflect HPV copy number in their tumor, and have prognosticimplications beyond disease stage and smoking history.

Attention was next turned to the HPV16 negative patients, and digitalPCR assays were developed for the 4 most common alternative HPV strains.This analysis is shown in FIG. 12. Indeed among the non-heavy smokers,all of the HPV16 negative patients had detectable HPV 18/31/33 or 35 intheir plasma. Among patients who were heavy smokers a significantlydifferent pattern was observed. Slightly fewer patients were HPV16positive. But among the HPV16 negative patients, only about 1/3rd hadvariant HPVs detectable, with the remainder negative for all 5 HPVstrains. Perhaps even more concerning, 2 out of these four patients hada positive neck dissection post-treatment, and one patient also testedpositive for a p53 mutation in the tumor. Among non-smokers no increaseddisease events with alternative HPV strains were observed, however,among smokers there were only 2 patients here but one of them developedregional recurrence soon after completing CRT. Thus, there seems to bean interactive effect of HPV16 negativity and smoking status.

Variable clearance kinetics among patients who were high expressers ofHPV16 was also observed. As shown in FIG. 13, some patients hadexponential clearance of HPV16 DNA and others had a more complex kineticpattern with delayed clearance from plasma. This was quantified bymeasuring how much plasma HPV DNA was present at week 4 relative to thepeak HPV value. For the top patient, this value is 0%, and for thebottom it is 39%. The median value of 5% was used to stratify patientsas having either rapid clearance or delayed clearance kinetics.

Putting this all together, plasma circulating tumor HPV16 based riskgroups were defined. This is depicted in FIG. 14. The favorable riskpatients had abundant HPV16 and rapid clearance kinetics. All otherpatients had unfavorable circulating tumor HPV16 profiles either due todelayed kinetics, low copy number, due to being positive for variantHPVs, or due to undetectable HPV. For both non-smokers and smokersapproximately 30% of patients had a favorable circulating tumor HPV16profile, and 70% were unfavorable. Again noted here is the 20% subset ofsmokers in whom the 5 most common HPV strains were not detected.

As shown in FIG. 4, patients with a favorable circulating tumor HPV16profile did not have any regional disease events—regardless of smokingstatus. However, in cases of patients having an unfavorable circulatingtumor HPV profile, smoking status had a major impact. Non-heavy smokersstill did well, with 90% regional disease control. However, heavysmokers or the minority presenting with T4 disease had dismal regionaldisease control when they also had unfavorable plasma circulating tumorHPV16 profiles.

In summary, plasma ctHPV16 DNA reveals genetic heterogeneity amongHPV-associated OPSCC patients 4/18 (22%) patients with >10 pack-yearstobacco or T4 disease and p16+ OPSCC were negative forHPV-16/18/31/33/35. Approximately 30% of HPV-associated OPSCC have afavorable ctHPV16 Profile (i.e., ≥200 copies/mL and rapid clearancekinetics) unfavorable ctHPV16 profiles (i.e., low/undetectable ordelayed clearance) are strongly associated with regional disease failurein heavy smokers. Assessment of ctHPV16 profiles can help guidetreatment intensity in non-smokers and smokers being treated forHPV-associated OPSCC.

Example 6: Application to Cancer Surveillance in a Longitudinal ClinicalStudy of Patients that Do Not Exhibit Any Clinical Evidence of CancerFollowing Therapy

A prospective study was conducted of 73 patients who previously werediagnosed with an HPV+ malignancy but were deemed to have “no evidenceof disease” after curative intent therapy. The HPV blood test wasapplied to these patients during a routine follow-up. None of thepatients who tested negative in the HPV blood test developed a new HPV+cancer or recurrence of the prior HPV+ cancer. In contrast, 9 out of 13patients who tested positive in the HPV blood test were diagnosed withan HPV+ cancer.

FIG. 7 depicts results of HPV blood tests. Negative samples can beclearly distinguished from positive samples. FIG. 8 shows a case summaryfor a patient who tested positive according to the HPV blood test, butwho exhibited no abnormality or evidence of disease at a follow-up visitwith the oncologist, and who had a recurrence of disease several monthsafter the follow-up.

A summary of the clinical results is shown in FIG. 7. These results showthe predictive effectiveness of the HPV blood test. Of the 73 patientsin the study, 60 tested negative according to the HPV blood test, and ofwhich none exhibited a recurrence of disease. In contrast, of the 13patients that tested positive according to the HPV blood test, 9 had arecurrence of disease, while the remaining 4 are being monitored closelyfor recurrence.

Example 7: Modified Oligonucleotide Compositions and Methods forDetection of Tumor-Derived HPV16

Provided in Table 14 below are primers and probes for detection oftumor-derived HPV16, where the “+” signifies a locked nucleic acid.Tumor derived HPV16 DNA can be detected and distinguished from non-tumorsources using any three primer/probe sets from Table 14 within a singlemultiplex digital PCR reaction, where the central probe has a detectioncolor distinct from both of the other (boundary) probes. The twoboundary probes may or may not have the same color. The DNA of theprimer/probe sets is modified to include quenchers, dye moieties andlocked nucleic acids. In this example, droplets that are positive forthe central probe's detection label and negative for the boundary/distalprobes detection label contain tumor derived viral DNA.

FIG. 9 depicts the results of a multiplexed digital PCR reaction todetect tumor-derived HPV16 DNA in a patient blood sample that includesmodified primer probe sets 3, 5 and 7 from Table 14, where detectionprobe 5 is conjugated to HEX and detection probes 3 and 7 are conjugatedto FAM.

As another example, modified primer probe sets 4, 7, and 10 from Table14 could be used for the multiplexed digital PCR reaction to detecttumor-derived HPV16 DNA, where detection probe 7 is conjugated to FAMand detection probes 4 and 10 are conjugated to HEX. Alternatively,detection 7 could be conjugated to FAM, probe 4 conjugated to HEX, andprobe 10 conjugated to a different detection moiety.

In some embodiments, the primer/probe sets may be selected so that notwo sets are consecutive in Table 14. For illustration, examples oftriads in this embodiment with no consecutive primer probe sets include{1, 3, 5}, {1, 3, 8}, {4, 6, 9}, . . . {14, 16, 18}. Examples of triadswith a consecutive primer probe set include {1, 2, 5} and {10, 12, 13}.

For the purposes of this application, digital PCR refers to any methodthat will be understood to those versed in the art where PCR reactionsare partitioned into many sub-reactions for analysis. The partitioncould be droplets or microwells or any other partition used for digitalPCR.

It is to be understood that HEX and FAM are used solely for clarity andillustrative purposes in all the examples.

TABLE 14 Primers and Probes for HPV16 Amplicon (size) Named Set (Assay)Detail as Sequence T_(M) ¹  1 1A Forward 1A_Primer15′TGC ACC AAA AGA GA+A CT3′ 60.4 (70 bp) primer (SEQ ID NO: 23) Reverse1A_Primer2 5′GTG CAT AAC TGT GGT +AAC 60.5 primer T3′(SEQ ID NO: 24)TaqMan® 1A_Probe 5′/Dye/CT+G +TG+G GTC +CT+G 68.2 probeA/Quencher/3′(SEQ ID NO: 25)  2 2A Forward 2A_Primer15′AGA GCT GCA AAC AAC TAT ACA3′ 62 (78 bp) primer (SEQ ID NO: 26) (E6)Reverse 2A_Primer2 5′TAT ACC TCA CGT CGC AGT3′ 62.2 primer(SEQ ID NO: 27) TaqMan® 2A_Probe 5′/Dye/AGA ATG TGT/Quencher- 67.5 probe2/GTA CTG CAA GCA ACA GTT/Quencher-1/3′ (SEQ ID NO: 28)  3 3A Forward3A_Primer1 5′ CGT GAG GTA TAT GAC TTT GCT 62.4 (75 bp) primerT 3′ (SEQ ID NO: 29) Reverse 3A_Primer2 5′ TCA CAT ACA GCA TAT GGA TTC62.1 primer C 3′ (SEQ ID NO: 30) TaqMan® 3A_Probe5′/Dye/CG+G +GAT +TTA +T+G+C 69.4 probe A/Quencher/3′ (SEQ ID NO: 31)  44A Forward 4A_Primer1 5′GTT TTA TTC TAA AAT TAG TGA+ 60.2 (75 bp) primerG+TA T3′ (SEQ ID NO: 32) Reverse 4A_Primer25′TTG TAT TGC TGT TCT A+AT GTT3′ 59.9 primer (SEQ ID NO: 33) TaqMan®4A_Probe 5′/Dye/AGT T+T+G +TA+T+ 67.1 probe G+GA/Quencher/3′(SEQ ID NO: 34)  5 5A Forward 5A_Primer15′ CAA ACC GTT GTG TGA TTT GT 3′ 61.9 (75 bp) primer (SEQ ID NO: 35)Reverse 5A_Primer2 5′ GAT GTC TTT GCT TTT CTT CAG 62 primerG 3′ (SEQ ID NO: 36) TaqMan® 5A_Probe 5′/Dye/AAG C+C+A +CTG+ 68.1 probeT+GT/Quencher/3′ (SEQ ID NO: 37)  6 6A Forward 6A_Primer1GGA CAA AAA GCA A+AG3′ 60.6 (75 bp) primer (SEQ ID NO: 38) Reverse6A_Primer2 5′GA+T GAT CTG CAA CAA GAC3′ 60.3 primer (SEQ ID NO: 39)TaqMan® 6A_Probe 5′/Dye/TCC AC+C G+A+C CCCT/ 68 probe Quencher/3′(SEQ ID NO: 40)  7 7A Forward 7A_Primer1 5′ TGC ATG AAT ATA TGT TAG ATT61.9 (76 bp) primer TGC A 3′ (SEQ ID NO: 41) Reverse 7A_Primer25′ TCC TCT GAG CTG TCA TTT AAT 61.9 primer T 3′ (SEQ ID NO: 42) TaqMan®7A_Probe 5′/Dye/A+C+A A+C+T +GAT CT+CT/ 68.2 probeQuencher/3′ (SEQ ID NO: 43)  8 8A Forward 8A_Primer15′TGA +AAT AGA TGG TCC AGC3′ 60 (74bp) primer (SEQ ID NO: 44) Reverse8A_Primer2 5′TGC AAC AAA AGG TTA CA+A 1A3′ 60 primer (SEQ ID NO: 45)TaqMan® 8A_Probe 5′/Dye/CAG +A+GC C+C+A T+T+A 68.1 probeC/Quencher/3′ (SEQ ID NO: 46)  9 9A Forward 9A_Primer15′TGA CTC TAC GCT TCG GTT G3′ 63.6 (73bp) primer (SEQ ID NO: 47) (E7)Reverse 9A_Primer2 5′GCC CAT TAA CAG GTC TTC C3′ 62.3 primer(SEQ ID NO: 48) TaqMan® 9A_Probe_v1 5′/Dye/TACAAAGCA/Quencher-2/CAC 68probe ACG TAG ACA TTC GTA CTT/Quencher-1/3′ (SEQ ID NO: 49) 9A_Probe_v25′/Dye/CGT ACA AAG/Quencher- 68.3 2/CAC ACA CGT AGA CAT TCGTAC/Quencher-1/3′ (SEQ ID NO: 50) 9A_Probe_v35′/Dye/CA+CA+C+G+TA+G+ACAT/ 67.7 Quencher/3′ (SEQ ID NO: 51) 10 1B Forward 1B_Primer1 5′ACT GCA ATG T+TT CAG G+A3′ 60.2 (79 bp) primer(SEQ ID NO: 52) Reverse 1B_Primer2 5′CAT +GTA +TAG +TTG ITT +GC3′ 60.1primer (SEQ ID NO: 53) TaqMan® 1B_Probe 5′/Dye/CGA CCC AGA/Quencher-68.3 probe 2/AAG TTA CCA CAG TTA TGC A/Quencher-1/3′ (SEQ ID NO: 54) 112B Forward 2B_Primer1 5′TTA GAA TGT GIG TAC TGC +AA3′ 60.2 (75 bp)primer (SEQ ID NO: 55) Reverse 2B_Primer2 5′CAT AAA TCC +CGA AAA GCA3′60.2 primer (SEQ ID NO: 56) TaqMan® 2B_Probe5′/Dye/GCA ACA GTT/Quencher- 68.3 probe 2/ACT GCG ACG TGA GGTAT/Quencher-1/3′ (SEQ ID NO: 57) 12 3B  Forward 3B_Primer15′CAT A+GT ATA T+AG AG+A TGG 60.1 (75 bp) primer GA3′ (SEQ ID NO: 58)Reverse 3B_Primer2 5′TC+T ATA C+TC A+CT AAT TT+T 60.3 primerAG3′ (SEQ ID NO: 59) TaqMan® 3B_Probe 5′/Dye/A+T+C A+C+A TA+C+ 66.6probe AGC/Quencher/3′ (SEQ ID NO: 60) 13 4B  Forward 4B_Primer15′CAT TAT TGT TAT A+G+T T+T+G 59.9 (73 bp) primer TA3′ (SEQ ID NO: 61)5′TAA TTA ACA AAT CAC A+CA ACG3′ (SEQ ID NO: 62)  Reverse 4B_Primer25′/Dye/TA+T +G+G+A A+CA A+CA 59.9 primer T/Quencher/3′ (SEQ ID NO: 63)TaqMan® 4B_Probe 5′TG+T ATT AA+C T+GT C+AA AAG3′ 67.6 probe(SEQ ID NO: 64) 14 5B Forward 5B_Primer1 5′GGA +ATC TTT GCT -ITT +TGT3′60.2 (70 bp) primer (SEQ ID NO: 65) Reverse 5B_Primer25′/Dye/TG+T +GT+C +CT+G AAG 60.1 primer A/Quencher/3′ (SEQ ID NO: 66)TaqMan® 5B_Probe 5′ATA ATA TAA GGG GTC G+GT G3′ 68 probe (SEQ ID NO: 67)15 6B Forward 6B_Primer1 5′AGC TGG GTT TCT CTA CG3′ 60.1 (80 bp) primer(SEQ ID NO: 68) Reverse 6B_Primer2 5′/Dye/CGG TCG ATG/Quencher- 60.4primer 2/TAT GTC TTG TTG CAG ATC ATC/Quencher-1/3′ (SEQ ID NO: 69)TaqMan® 6B_Probe_v1 5′/Dye/CCG GTC GAT/Quencher- 68.4 probe2/GTA TGT CTT GTT GCA GAT/Quencher-1/3′ (SEQ ID NO: 70) 6B_Probe_v25′TGC ATG GAG ATA CAC CT3′ 68.3 (SEQ ID NO: 71) 16 7B Forward 7B_Primer15′ACA GTA G+AG ATC AGT TGT3′ 59.7 (72 bp) primer (SEQ ID NO: 72) Reverse7B_Primer2 5′/Dye/CC+A G+A+G A+C+A 59.9 primer A+CT/Quencher/3′(SEQ ID NO: 73) TaqMan® 7B_Probe 5′TAT GAG CAA TTA A+A+T G+AC 68.4 probeA3′ (SEQ ID NO: 74) 17 8B Forward 8B_Primer15′ATT GT+A ATG GGC TCT GTC3′ 60 (92 bp) primer (SEQ ID NO: 75) Reverse8B_Primer2 5′/Dye/ATT T+C+A TC+C +T+C+C 59.9 primer T/Quencher/3′(SEQ ID NO: 76) TaqMan® 8B_Probe 5′TAC +AAA GCA CAC ACG TAG3′ 68.1 probe(SEQ ID NO: 77) 18 9B Forward 9B_Primer1 5′TT+A TGG ITT CTG AGA ACA 60(92 bp) primer GAT3′ (SEQ ID NO: 78) Reverse 9B_Primer25′/Dye/TGG AAG ACC/Quencher- 60.4 primer 2/TGT TAA TGG GCA CAC TaqMan®9B_Probe T/Quencher-1/3′ 68.1 probe (SEQ ID NO: 79) ¹″Dye″ stands for afluorescent reporter dye. Any reporter dyes such as FAM™, HEX™, VIC®,Cy5™, and Cy5.5™ that is compatible with the detection channels for theinstrument can be used. A quencher compatible with the respective dyewas used at 3′ end of the probe (Quencher-1). A second quencher(Quencher-2) such as ZEN was sometimes placed internally between 9^(th)and 10^(th) nucleotide base from the reporter dye to enhance the overalldye quenching. ² These estimates of melting temperature(T_(M)) werecalculated using IDT′S oligo analyzer tools with following settings: (a)For primers: Target type = DNA, Oligo concentration = 0.9 μM, Na⁺concentration = 50 mM, Mg⁺⁺ concentration = 3 mM, and dNTPsconcentration = 0.8 mM. (b) for Probe: Target type = DNA, Oligoconcentration = 0.25 μM, Na⁺ concentration = 50 mM, Mg⁺⁺ concentration= 3 mM, and dNTPs concentration = 0.8 mM. ³ + = Locked nucleic acid

Example 8: Modified Oligonucleotide Compositions and Methods forDetection of Tumor-Derived HPV18

Provided in Table 15 below are primers and probes for detection oftumor-derived HPV18, where the “+” signifies a locked nucleic acid.Tumor derived HPV18 DNA can be detected and distinguished from non-tumorsources using any three primer/probe sets from Table 15 within a singlemultiplex digital PCR reaction, where the central probe has a detectioncolor distinct from both of the other (boundary) probes. The twoboundary probes may or may not have the same color. The DNA of theprimer/probe sets is modified to include quenchers, dye moieties, andlocked nucleic acids. In this example, droplets that are positive forthe central probe's detection label and negative for the boundary/distalprobes detection label contain tumor derived viral DNA.

For example, modified primer probe sets 4, 7, and 10 from Table 15 couldbe used for the multiplexed digital PCR reaction to detect tumor-derivedHPV18 DNA, where detection probe 7 is conjugated to FAM and detectionprobes 4 and 10 are conjugated to HEX. Alternatively, detection 7 couldbe conjugated to FAM, probe 4 conjugated to HEX, and probe 10 conjugatedto a different detection moiety.

In some embodiments, the primer/probe sets may be selected so that notwo sets are consecutive in Table 15. For illustration, examples oftriads in this embodiment with no consecutive primer probe sets include{1, 3, 5}, {1, 3, 8}, {4, 6, 9}, . . . {14, 16, 18}. Examples of triadswith a consecutive primer probe set include {1, 2, 5} and {10, 12, 13}.

For the purposes of this application, digital PCR refers to any methodthat will be understood to those versed in the art where PCR reactionsare partitioned into many sub-reactions for analysis. The partitioncould be droplets or microwells or any other partition used for digitalPCR.

It is to be understood that HEX and FAM are used solely for clarity andillustrative purposes in all the examples.

TABLE 15 Primers and probes for HPV18 Amplicon (size) Set (Assay) DetailNamed as Sequence T_(M) ¹ 1 1C Forward primer 1C_Primer15′CGC TTT GAG GAT CCA AC3′ 60 (70 bp) (SEQ ID NO: 80) (E6)Reverse primer 1C_Primer2 5′GCA GTG AAG TGT TCA GTT3′ 60 (SEQ ID NO: 81)TaqMan® 1C_Probe 5′/Dye/ACC CTA CAA/Quencher-2/GCT ACC TGA 67.6 probeTCT GTG C/Quencher-1/3′ (SEQ ID NO: 82) 2 2C Forward primer 2C_Primer15′AAG ACA +TAG AAA TAA CCT +GT3′ 60.2 (76 bp) (SEQ ID NO: 83) (E6)Reverse primer 2C_Primer2 5′TTA A+A+T +G+CA AAT TCA AAT3′ 60.3(SEQ ID NO: 84) TaqMan® 2C_Probe5′/Dye/TGC AAG ACA /Quencher-2/GTA TTG GAA 67.7 probeCTT ACA GAG GT/Quencher-1/3′ (SEQ ID NO: 85) 3 3C Forward primer3C_Primer1 5′TTA T+TT GTG GTG TAT A+GA GA3′ 59.8 (80 bp) (SEQ ID NO: 86)(E6) Reverse primer 3C_Primer2 5′AAT T+CT +CTA ATT +CTA GAA TAA3′ 59.8(SEQ ID NO: 87) TaqMan probe 3C_Probe5′/Dye/CA+T G+C+G +G+TA +TAC T/Quencher/3′ 68.2 (SEQ ID NO: 88) 4 4CForward primer 4C_Primer1 5′AAG AC+A TTA +TTC AGA C+TC T3′ 60.2 (72 bp)(SEQ ID NO: 89) (E6) Reverse primer 4C_Primer25′AAT AAA TTG +TAT AAC +C+CA3′ 60.4 (SEQ ID NO: 90) TaqMan® 4C_Probe5′/Dye/TG+G +AGA +C+A+C +ATT/Quencher/3′ 67.6 probe (SEQ ID NO: 91) 5 5CForward primer 5C_Primer1 5′AGAAACCGTT G AATCCAG3′ 59.5 (71 bp)(SEQ ID NO: 92) (E6) Reverse primer 5C_Primer25′ATT TCA CAA CAT AGC TGG G3′ 60.1 (SEQ ID NO: 93) TaqMan® 5C_Probe5′/Dye/AG+A CA+C +C+TT AA+T +GA/Quencher/3′ 68.1 probe (SEQ ID NO: 94) 66C Forward primer 6C_Primer1 5′CCA GTG CCA TTC GTG C3′ 60.8 (80 bp)(SEQ ID NO: 95) (E6) Reverse primer 6C_Primer25′TTA TA+C TTG +TGT TTC TCT G3′ 60.1 (SEQ ID NO: 96) TaqMan® 6C_Probe5′/Dye/CAC GAC AGG /Quencher-2/AAC GAC 68.3 probeTCC AAC GAC/Quencher-1/3′ (SEQ ID NO: 97) 7 7C Forward primer 7C_Primer15′ATG GAC CTA AGG CAA CA3′ 60.3 (72 bp) (SEQ ID NO: 98) (E7)Reverse primer 7C_Primer2 5′TG+A CAT AGA AGG TCA ACC3′ 60.2(SEQ ID NO: 99) TaqMan® 7C_Probe 5′/Dye/TT+T A+G+A +G+CC +CC/Quencher/3′68.2 probe (SEQ ID NO: 100) 8 8C Forward primer 8C_Primer15′CGA GCA A+TT AAG CGA CTC3′ 60.2 (75 bp) (SEQ ID NO: 101) (E7)Reverse primer 8C_Primer2 5′CGG GCT GGT AAA TGT TGA3′ 60(SEQ ID NO: 102) TaqMan® 8C_Probe5′/Dye/T+C+G +TTT T+CT T+C+C T/Quencher/3′ 68.1 probe (SEQ ID NO: 103) 99C(75 bp) Forward primer 9C_Primer1 5′ACA ACG TCA CAC AAT GTT3′ 60.1(E7) (SEQ ID NO: 104) Reverse primer 9C_Primer25′TCT GCT GG CTT TCT ACT3′ 60 (SEQ ID NO: 105) TaqMan® 9C_Probe5′/Dye/TGT ATG TGT /Quencher-2/TGT AAG TGT 66.9 probeGAA GCC AGA AT/Quencher-1/3′ (SEQ ID NO: 106) 10 10C Forward primer10C_Primer1 5′ACC TTC GAG CAT TCC A3′ 60.2 (74 bp) (SEQ ID NO: 107) (E7)Reverse primer 10C_Primer2 5TTACTGCTGGGATGCA3′ 60.2 (SEQ ID NO: 108)TaqMan® 10C_Probe 5′/Dye/CTG AA+C +AC+C C+T+G T/Quencher/3′ 68.2 probe(SEQ ID NO: 109) 11 1D Forward primer 1D_Primer15′CCC TAC AAG CTA CCT GAT3′ 60 (83 bp) (SEQ ID NO: 110) (E6)Reverse primer 1D_Primer2 5′CAA TAT A+CA +CAG +GT+T ATT T3′ 60.1(SEQ ID NO: 111) TaqMan® 1D_Probe5′/Dye/CA+C +G+GA A+C+T GAA C/Quencher/3′ 68.1 probe (SEQ ID NO: 112) 122D Forward primer 2D_Primer1 5TAT TT+G +AAT TT+G +CAT +TTA3′ 59.9(92 bp) (SEQ ID NO: 113) (E6) Reverse primer 2D_Primer25′AAT +CTA TA+C +ATT TAT +GGC3′ 59.9 (SEQ ID NO: 114) TaqMan® 2D_Probe5′/Dye/AG+A C+AG +TA+T AC+C 67.9 probe +GC/Quencher/3′ (SEQ ID NO: 115)13 3D Forward primer 3D_Primer1 5′CTA G+AA TTA +G+A+G AAT T+AA GA3′ 60.1(74 bp) (SEQ ID NO: 116) (E6) Reverse primer 3D_Primer25′AGT GTT AGT TAG TTT +TTC CAA T3′ 60.1 (SEQ ID NO: 117) TaqMan®3D_Probe 5′/Dye/TC+A +G+A+C T+CT G+TG T/Quencher/3′ 68.4 probe(SEQ ID NO: 118) 14 4D Forward primer 4D_Primer15′ATT A+AT AAG GTG CC+T GC3′ 60.2 (75 bp) (SEQ ID NO: 119) (E6)Reverse primer 4D_Primer2 5′TCG TTT TTC ATT A+AG GTG T3′ 60(SEQ ID NO: 120) TaqMan® 4D_Probe5′/Dye/TGA AT+C +C+AG C+A+G A/Quencher/3′ 67.8 probe (SEQ ID NO: 121) 155D Forward primer 5D_Primer1 5′ATT TCA CAA CAT AGC TGG G3′ 60.1 (98 bp)(SEQ ID NO: 122) (E6) Reverse primer 5D_Primer25′GTT TCT CTG CGT CGT TG3′ 60.4 (SEQ ID NO: 123) TaqMan® 5D_Probe5′/Dye/AG+T G+C+C +A+TT C+GT G/Quencher/3′ 68.3 probe (SEQ ID NO: 124)16 6D Forward primer 6D_Primer1 5′ATT +GTA TTG CAT TTA GAG CC3′ 59.8(90 bp) (SEQ ID NO: 125) (E7) Reverse primer 6D_Primer25′TTC ATC GTT TTC TTC +CTC3′ 59.9 (SEQ ID NO: 126) TaqMan® 6D_Probe5′/Dye/CTA T+G+T +CA+C +G+AG C/Quencher/3′ 68.2 probe (SEQ ID NO: 127)17 7D Forward primer 7D_Primer1 5′ATA GAT G+GA GTT A+AT CAT CA3′ 59.7(82 bp) (SEQ ID NO: 128) (E7) Reverse primer 7D_Primer25′AC+T TAC AAC ACA +TAC ACA3′ 60 (SEQ ID NO: 129) TaqMan® 7D_Probe5′/Dye/CCG AAC CAC /Quencher-2/AAC GTC ACA 67.8 probeCAA TGT T/Quencher-1/3′ (SEQ ID NO: 130) 18 8D Forward primer 8D_Primer15′TGA AGC CAG AAT TGA GCT AG3′ 61.9 (83 bp) (SEQ ID NO: 131) (E7)Reverse primer 8D_Primer2 5′AGG ACA GGG TGT TCA GAA3′ 62.6(SEQ ID NO: 132) TaqMan® 8D_Probe 5′/Dye/CA+GA+C+GAC+CTTCG/Quencher/3′65.9 probe (SEQ ID NO: 133) ¹“Dye” stands for a fluorescent reporterdye. Any reporter dyes such as FAM™, HEX™, VIC®, Cy5™, and Cy5.5™ thatis compatible with the detection channels for the instrument can beused. A quencher compatible with the respective dye was used at 3′ endof the probe (Quencher-1). A second quencher (Quencher-2) such as ZENwas sometimes placed internally between 9^(th) and 10^(th) nucleotidebase from the reporter dye to enhance the overall dye quenching. ²Theseestimates of melting temperature(T_(M)) were calculated using IDT′Soligo analyzer tools with following settings: (a) For primers: Targettype = DNA, Oligo concentration = 0.9 μM, Na⁺ concentration = 50 mM,Mg⁺⁺ concentration = 3 mM, and dNTPs concentration = 0.8 mM. (b) forProbe: Target type = DNA, Oligo concentration = 0.25 μM, Na⁺concentration = 50 mM, Mg⁺⁺ concentration = 3 mM, and dNTPsconcentration = 0.8 mM. ³+ = Locked nucleic acid

Example 9: Modified Oligonucleotide Compositions and Methods forDetection of Tumor-Derived HPV31

Provided in Table 16 below are primers and probes for detection oftumor-derived HPV31, where the “+” signifies a locked nucleic acid.Tumor derived HPV31 DNA can be detected and distinguished from non-tumorsources using any three primer/probe sets from Table 16 within a singlemultiplex digital PCR reaction, where the central probe has a detectioncolor distinct from both of the other (boundary) probes. The twoboundary probes may or may not have the same color. The DNA of theprimer/probe sets is modified to include quenchers, dye moieties andlocked nucleic acids. In this example, droplets that are positive forthe central probe's detection label and negative for the boundary/distalprobes detection label contain tumor derived viral DNA.

For example, modified primer probe sets 4, 7, and 10 from Table 16 couldbe used for the multiplexed digital PCR reaction to detect tumor-derivedHPV31 DNA, where detection probe 7 is conjugated to FAM and detectionprobes 4 and 10 are conjugated to HEX. Alternatively, detection 7 couldbe conjugated to FAM, probe 4 conjugated to HEX, and probe 10 conjugatedto a different detection moiety.

In some embodiments, the primer/probe sets may be selected so that notwo sets are consecutive in Table 16. For illustration, examples oftriads in this embodiment with no consecutive primer probe sets include{1, 3, 5}, {1, 3, 8}, {4, 6, 9}, . . . {13, 15, 17}. Examples of triadswith a consecutive primer probe set include {1, 2, 5} and {10, 12, 13}.

For the purposes of this application, digital PCR refers to any methodthat will be understood to those versed in the art where PCR reactionsare partitioned into many sub-reactions for analysis. The partitioncould be droplets or microwells or any other partition used for digitalPCR.

It is to be understood that HEX and FAM are used solely for clarity andillustrative purposes in all the examples.

TABLE 16 Primers and probes for HPV31 Amplicon (size) Set (Assay) DetailNamed as Sequence T_(M) ¹ 1 1E Forward primer 1E_Primer15′ATG TTC AAA AA+T CCT GCA3′ 60 (75 bp) (SEQ ID NO: 134) (E6)Reverse primer 1E_Primer2 5′TTC ATC GTA GGG TAT +TTC C3′ 60.2(SEQ ID NO: 135) TaqMan® probe 1E_Probe5′/Dye/AAC +T+AA G+C+T C+G+G C/Quencher/3′ 68.3 (SEQ ID NO: 136) 2 2EForward primer 2E_Primer1 5′CT+A AGA TTG A+AT TGT GT+C T3′ 59.9 (75 bp)(SEQ ID NO: 137) (E6) Reverse primer 2E_Primer25′TAA ATC +TGT AAA TGC AA+A ATC TA 3′ 59.9 (SEQ ID NO: 138)TaqMan® probe 2E_Probe 5′/Dye/AC+A GA+A A+CA +G+AG 68.1 +GT/Quencher/3′(SEQ ID NO: 139) 3 3E Forward primer 3E_Primer15′ACA ATA +GTA TAT AGG GAC GAC3′ 59.9 (75 bp) (SEQ ID NO: 140) (E6)Reverse primer 3E_Primer2 5′TTC ACT +TAC TTT TGA +ATA +AAA T3′ 59.8(SEQ ID NO: 141) TaqMan® probe 3E_Probe5′/Dye/AC+G +G+AG TG+T +GTA C/Quencher/3′ 68.1 (SEQ ID NO: 142) 4 4EForward primer 4E_Primer1 5′TTT A+GA TG+G TAT AGA TAT AGT GT3′ 60(81 bp) (SEQ ID NO: 143) (E6) Reverse primer 4E_Primer25′CC+T AAT TAA +C+AA ATC ACA TA3′ 60 (SEQ ID NO: 144) TaqMan® probe4E_Probe 5′/Dye/A+T+G +G+AA +C+AA CAT T/Quencher/3′ 67.7(SEQ ID NO: 145) 5 5E Forward primer 5E_Primer15′TGT AT+A ACG TGT +CAA AGA C 60 (74 bp) (SEQ ID NO: 146) (E6)Reverse primer 5E_Primer2 5′TTG TGG AAT C+GT T+TC TTT3′ 59.9(SEQ ID NO: 147) TaqMan® probe 5E_Probe5′/Dye/TGT +G+TC +C+AG A+A+G A/Quencher/3′ 68.4 (SEQ ID NO: 148) 6 6EForward primer 6E_Primer1 CAT AGG AGG AAG GTG GAC 60.5 (70 bp)(SEQ ID NO: 149) (E6) Reverse primer 6E_Primer25′TTA CAC TT+G GGT +TTC AG3′ 60.2 (SEQ ID NO: 150) TaqMan® probe6E_Probe 5′/Dye/CGT TGC ATA /Quencher-2/GCA TGT 67.4TGG AGA AGA CC/Quencher-1/3′ (SEQ ID NO: 151) 7 7E Forward primer7E_Primer1 5′CAA GA+C TAT GTG TTA +GAT T3′ 59.9 (75 bp) (SEQ ID NO: 152)(E7) Reverse primer 7E_Primer2 5′TCA TCT GAG CTG TC+G G+GT AAT T3′ 60.1(SEQ ID NO: 153) TaqMan® probe 7E_Probe5′/Dye/AAC TGA +C+C+T C+C+A C/Quencher/3′ 68.3 (SEQ ID NO: 154) 8 8EForward primer 8E_Primer1 5′AGG ATG TCA TA+G ACA GTC3′ 59.8 (89 bp)(SEQ ID NO: 155) (E7) Reverse primer 8E_Primer25′TG+T AGA CTT ACA CTG ACA A3′ 60.3 (SEQ ID NO: 156) TaqMan® probe8E_Probe 5′/Dye/CTG +G+AC A+AG +C+AG A/Quencher/3′ 68 (SEQ ID NO: 157) 99E Forward primer 9E_Primer1 5′AGC ACA CAA GTA GAT ATT CGC3′ 62.4(79 bp) (SEQ ID NO: 158) (E7) Reverse primer 9E_Primer25′TAG TAG AAC AGT TGG GGC A3′ 62.6 (SEQ ID NO: 159) TaqMan® probe9E_Probe 5′/Dye/TAA+C+AG+CT+C+TTG+C/Quencher/3′ 65.3 (SEQ ID NO: 160) 101F Forward primer 1F_Primer1 5′AAA TTG CAT G+AA CTA +AGC3′ 60.2 (82 bp)(SEQ ID NO: 161) (E6) Reverse primer 1F_Primer25′TT+A ACT GAC CTT TGC AGT3′ 59.8 (SEQ ID NO: 162) TaqMan® probe1F_Probe 5′/Dye/CGG CAT TGG /Quencher-2/AAA TAC 68.1CCT ACG ATG AAC TAA/Quencher-1/3′ (SEQ ID NO: 163) 11 2F Forward primer2F_Primer1 5′CAG AA+A CAG AGG TAT TA+G AT3′ 60.1 (90 bp)(SEQ ID NO: 164) (E6) Reverse primer 2F_Primer25′TTA +AAC ATT TTG TAC A+CA CT3′ 59.8 (SEQ ID NO: 165) TaqMan® probe2F_Probe 5′/Dye/AC+G +A+CA +C+AC CAC A/Quencher/3′ 68.1 (SEQ ID NO: 166)12 3F Forward primer 3F_Primer1 5′ATT T+TA TTC AA+A AGT A+AG TGA3′ 59.8(81 bp) (SEQ ID NO: 167) (E6) Reverse primer 3F_Primer25′CCT +TTG TTT GTC +AAT T+TT TC3′ 60.1 (SEQ ID NO: 168) TaqMan® probe3F_Probe 5′/Dye/TAG +T+G+T GTA +T+G+G A/Quencher/3′ 68.7(SEQ ID NO: 169) 13 4F Forward primer 4F_Primer15′TAT ATG TGA TTT GTT A+AT TA+G GT3′ 60 (75 bp) (SEQ ID NO: 170) (E6)Reverse primer 4F_Primer2 5′TCC AA+A TGT C+TT TGT TTT TC3′ 60.1(SEQ ID NO: 171) TaqMan® probe 4F_Probe5′/Dye/CC+G TT+G T+G+T +C+CA G/Quencher/3′ 68.1 (SEQ ID NO: 172) 14 5FForward primer 5F_Primer1 5′TAA AAA G+AA ACG ATT +CCA C3′ 60.1 (80 bp)(SEQ ID NO: 173) (E6) Reverse primer 5F_Primer25′TT+T CAG TAC GAG GTC TTC3′ (SEQ ID NO: 174) TaqMan® probe 5F_Probe5′/Dye/AAG GTG GAC /Quencher-2/AGG ACG 66.5 TTG CA/Quencher-1/3′(SEQ ID NO: 175) 15 6F Forward primer 6F_Primer15′TGG AGA AAC ACC TAC GT3′ 59.8 (74 bp) (SEQ ID NO: 176) (E7)Reverse primer 6F_Primer2 5′TAA TTG CTC AT+A ACA GTG GA3′ 60(SEQ ID NO: 177) TaqMan® probe 6F_Probe5′/Dye/AA+C +C+T+G AGG CAA C/Quencher/3′ 68.3 (SEQ ID NO: 178) 16 7FForward primer 7F_Primer1 5′AGA TGA GGA GGA TGT C+AT3′ 60.3 (74 bp)(SEQ ID NO: 179) (E7) Reverse primer 7F_Primer25′AG+G TAA +CGA TAT T+GT AAT T3′ 59.8 (SEQ ID NO: 180) TaqMan® probe7F_Probe 5′/Dye/CA+A +G+C+A G+AA C+CG/Quencher/3′ 67.9 (SEQ ID NO: 181)17 8F Forward primer 8F_Primer1 5′TGT CAG T+GT AAG TC+T ACA3′ 60.2(90 bp) (SEQ ID NO: 182) (E7) Reverse primer 8F_Primer25′TCC AAA TGA GCC C+AT TAA3′ 59.9 (SEQ ID NO: 183) TaqMan® probe8F_Probe 5′/Dye/TGT +A+C+A +GA+G CA+C A/Quencher/3′ 68.2(SEQ ID NO: 184) ¹“Dye” stands for a fluorescent reporter dye. Anyreporter dyes such as FAM™, HEX™, VIC®, Cy5™, and Cy5.5™ that iscompatible with the detection channels for the instrument can be used. Aquencher compatible with the respective dye was used at 3′ end of theprobe (Quencher-1). A second quencher (Quencher-2) such as ZEN wassometimes placed internally between 9^(th) and 10^(th) nucleotide basefrom the reporter dye to enhance the overall dye quenching. ² Theseestimates of melting temperature(TM) were calculated using IDT′S oligoanalyzer tools with following settings: (a) For primers: Target type= DNA, Oligo concentration = 9 μM, Na⁺ concentration = 50 mM, Mg⁺⁺concentration = 3 mM, and dNTPs concentration = 8 mM. (b) for Probe:Target type = DNA, Oligo concentration ³ + = Locked nucleic acid

Example 10: Modified Oligonucleotide Compositions and Methods forDetection of Tumor-Derived HPV33

Provided in Table 17 below are primers and probes for detection oftumor-derived HPV33, where the “+” signifies a locked nucleic acid.Tumor derived HPV33 DNA can be detected and distinguished from non-tumorsources using any three primer/probe sets from Table 17 within a singlemultiplex digital PCR reaction, where the central probe has a detectioncolor distinct from both of the other (boundary) probes. The twoboundary probes may or may not have the same color. The DNA of theprimer/probe sets is modified to include quenchers, dye moieties andlocked nucleic acids. In this example, droplets that are positive forthe central probe's detection label and negative for the boundary/distalprobes detection label contain tumor derived viral DNA.

For example, modified primer probe sets 4, 7, and 10 from Table 17 couldbe used for the multiplexed digital PCR reaction to detect tumor-derivedHPV33 DNA, where detection probe 7 is conjugated to FAM and detectionprobes 4 and 10 are conjugated to HEX. Alternatively, detection 7 couldbe conjugated to FAM, probe 4 conjugated to HEX, and probe 10 conjugatedto a different detection moiety.

In some embodiments, the primer/probe sets may be selected so that notwo sets are consecutive in Table 17. For illustration, examples oftriads in this embodiment with no consecutive primer probe sets include{1, 3, 5}, {1, 3, 8}, {4, 6, 9}, . . . {11, 13, 15}. Examples of triadswith a consecutive primer probe set include {1, 2, 5} and {10, 12, 13}.

For the purposes of this application, digital PCR refers to any methodthat will be understood to those versed in the art where PCR reactionsare partitioned into many sub-reactions for analysis. The partitioncould be droplets or microwells or any other partition used for digitalPCR.

It is to be understood that HEX and FAM are used solely for clarity andillustrative purposes in all the examples.

TABLE 17 Primers and probes for HPV33 Amplicon (size) Set (Assay) DetailNamed as Sequence T_(M) ¹ 1 1G (75 bp) Forward primer 1G_Primer15′TTC AAG ACA CTG AGG +AAA3′ 59.9 (E6 assay) (SEQ ID NO: 185)Reverse primer 1G_Primer2 5′AAT GTT GT+G TAT AGT T+GT CTC3′ 60.2(SEQ ID NO: 186) TaqMan® probe 1G_Probe5′/Dye/AAC ATT GCA /Quencher-2/TGA TTT 67.6GTG CCA AGC ATT/Quencher-1 /3′ (SEQ ID NO: 187) 2 2G (77 bp)Forward primer 2G_Primer1 5′AAT GCA AAA AAC CTT +TGC3′ 60.1 (E6 assay)(SEQ ID NO: 188) Reverse primer 2G_Primer25′TCC C+TC TCT +ATA TAC A+AC T3′ 60.2 (SEQ ID NO: 189) TaqMan® probe2G_Probe 5′/Dye/CG+A TC+T +G+AG G+T+A 67.1 T/Quencher/3′(SEQ ID NO: 190) 3 3G (83 bp) Forward primer 3G_Primer15′AAT +CCA TTT G+GA ATA TG+T A3′ 59.8 (E6 assay) (SEQ ID NO: 191)Reverse primer 3G_Primer2 5′CCA +TAT A+CA +GAA T+AA TTA TAA 60 TG3′(SEQ ID NO: 192) TaqMan® probe 3G_Probe 5′/Dye/CT+G +T+GT TTG +C+GG 68.2T/Quencher/3′ (SEQ ID NO: 193) 4 4G (75 bp) Forward primer 4G_Primer15′TGA +A+AT ATT AAT TA+G +GTG T3′ 60 (E6 assay) (SEQ ID NO: 194)Reverse primer 4G_Primer2 5′TTT AAA TCC ACA TGT +CGT T3′ 59.9(SEQ ID NO: 195) TaqMan® probe 4G_Probe 5′/Dye/TG+T GTC +C+T+C +AA+G 68A/Quencher/3′ (SEQ ID NO: 196) 5 5G (82 bp) Forward primer 5G_Primer15′CAA A+CG +ATT T+CA TAA TA+T T3′ 59.9 (E6 assay) (SEQ ID NO: 197)Reverse primer 5G_Primer2 5′CAG TGC AGT TTC TCT ACG3′ 59.6(SEQ ID NO: 198) TaqMan® probe 5G_Probe 5′/Dye/ACA +CG+C CGC +ACA 68.1G/Quencher/3′ (SEQ ID NO: 199) 6 6G (75 bp) Forward primer 6G_Primer15′ACA CAA GCC AAC GTT AAA3′ 60.1 (E6 assay) (SEQ ID NO: 200)Reverse primer 6G_Primer2 5′AAT TGC TCA TA+G CA+G TAT A3′ 59.9(SEQ ID NO: 201) TaqMan® probe 6G_Probe 5′/Dye/ATC +C+T+G AA+C +CAA 67.8C/Quencher/3′ (SEQ ID NO: 202) 7 7G (83 bp) Forward primer 7G_Primer15′AAG TGA CAG CTC AGA TGA3′ 60.3 (E7 assay) (SEQ ID NO: 203)Reverse primer 7G_Primer2 5′TTA CA+A TGT AGT AA+T CAG CT3′ 60.1(SEQ ID NO: 204) TaqMan® probe 7G_Probe 5′/Dye/CG+G T+C+C AA+G CCT 67.8T/Quencher/3′ (SEQ ID NO: 205) 8 8G (87 bp) Forward primer 8G_Primer15′TAACACC AC AGTT CGTTTATGT3′ 62.4 (E7 assay) (SEQ ID NO: 206)Reverse primer 8G_Primer2 5′ACAATATTCACTGTGCCCATA3′ 61.9(SEQ ID NO: 207) TaqMan® probe 8G_Probe_v 5′/Dye/TG +AC+C +TA+CG 66.4 1+A+ACC/Quencher/3′ (SEQ ID NO: 208) 8G_Probe_v5′/Dye/CAG +TA+C +AG+C A+A+G 68.3 2 T/Quencher/3′ (SEQ ID NO: 209) 91H (81 bp) Forward primer 1H_Primer1 5′CAA GCA TTG GAG AC+A ACT A3′ 60.3(E6 assay) (SEQ ID NO: 210) Reverse primer 1H_Primer25′TAT ACC TCA GAT CG+T TGC3′ 60 (SEQ ID NO: 211) TaqMan® probe 1H_Probe5′/Dye/TC+C A+C+G CAC +TG+T 68.6 A/Quencher/3′ (SEQ ID NO: 212) 102H (75 bp) Forward primer 2H_Primer1 5′TGA TT+T TGC ATT TGC AGA3′ 60.1(E6 assay) (SEQ ID NO: 213) Reverse primer 2H_Primer25′CAA A+CA CA+G TTT A+CA TAT3′ 60 (SEQ ID NO: 214) TaqMan® probe2H_Probe 5′/Dye/TT+C +C+CT +C+T+C TAT 68 A/Quencher/3′ (SEQ ID NO: 215)11 3H (79 bp) Forward primer 3H_Primer15′GTT +C+TT AT+C TAA AAT TA+G TG3′ 59.7 (E6 assay) (SEQ ID NO: 216)Reverse primer 3H_Primer2 5′TTT AAC TG+T TTG TTC +TAA TGT3′ 60(SEQ ID NO: 217) TaqMan® probe 3H_Probe_v 5′/Dye/TT+C +C+AT ATA +C+A+G65 1 A/Quencher/3′ (SEQ ID NO: 218) 3H_Probe_v5′/Dye/T+CT G+TA TA+T +G+G+A 65 2 A/Quencher/3′ (SEQ ID NO: 219) 124H (80 bp) Forward primer 4H_Primer1 5′AGG TGT ATT ATA T+GT CAA A+GA3′59.9 (E6 assay) (SEQ ID NO: 220) Reverse primer 4H_Primer25′ATA TTA TGA AAT +C+G+T TTG TT3′ 60.2 (SEQ ID NO: 221) TaqMan® probe4H_Probe_v 5′/Dye/TT+G T+GT +C+C+T CA+A 67.9 1 G/Quencher/3′(SEQ ID NO: 222) 4H_Probe_v 5′/Dye/CG+A +CAT GT+G +G+AT 67 2T/Quencher/3′ (SEQ ID NO: 223) 13 5H (74 bp) Forward primer 5H_Primer15′AG+G +AAT ATG T+TT TA+G ATT3′ 60.1 (E6 assay) (SEQ ID NO: 224)Reverse primer 5H_Primer2 5′ATC TGA GCT GTC ACT +TAA3′ 60.2(SEQ ID NO: 225) TaqMan® probe 5H_probe 5′/Dye/TG+A +C+C+T A+TA +CTG68.1 C/Quencher/3′ (SEQ ID NO: 226) 14 6H (77 bp) Forward primer6H_Primer1 5′GAG GAT GAA GGC TTG GA3′ 60.6 (E7 assay) (SEQ ID NO: 227)Reverse primer 6H_Primer2 5′TGA CA+A CAG GTT A+CA AT3′ 60.3(SEQ ID NO: 228) TaqMan® probe 6H_probe5′/Dye/CCA GAT GGA /Quencher-2/CAA 67.7 GCA CAA CCA GC/Quencher-1/3′(SEQ ID NO: 229) 15 7H (77 bp) Forward primer 7H_Primer15′TGT CA+A CAG TAC AGC AA3′ 60 (E7 assay) (SEQ ID NO: 230)Reverse primer 7H_Primer2 5′AGG TAG GGC ACA CAA TAT3′ 60.2(SEQ ID NO: 231) TaqMan® probe 7H_Probe 5′/Dye/AC+C +ATA +C+A+G +CAA68.1 C/Quencher/3′ (SEQ ID NO: 232) ¹“Dye” stands for a fluorescentreporter dye. Any reporter dyes such as FAM™, HEX™, VIC®, Cy5™, andCy5.5™ that is compatible with the detection channels for the instrumentcan be used. A quencher compatible with the respective dye was used at3′ end of the probe (Quencher-1). A second quencher (Quencher-2) such asZEN was sometimes placed internally between 9^(th) and 10^(th)nucleotide base from the reporter dye to enhance the overall dyequenching. ² These estimates of melting temperature(T_(M)) werecalculated using IDT′S oligo analyzer tools with following settings: (a)For primers: Target type = DNA, Oligo concentration = 9 μM, Na⁺concentration = 50 mM, Mg⁺⁺ concentration = 3 mM, and dNTPsconcentration = 8 mM. (b) for Probe: Target type = DNA, Oligoconcentration ³ + = Locked nucleic acid

Example 11: Modified Oligonucleotide Compositions and Methods forDetection of Tumor-Derived HPV35

Provided in Table 18 below are primers and probes for detection oftumor-derived HPV35, where the “+” signifies a locked nucleic acid.Tumor derived HPV35 DNA can be detected and distinguished from non-tumorsources using any three primer/probe sets from Table 18 within a singlemultiplex digital PCR reaction, where the central probe has a detectioncolor distinct from both of the other (boundary) probes. The twoboundary probes may or may not have the same color. The DNA of theprimer/probe sets is modified to include quenchers, dye moieties andlocked nucleic acids. In this example, droplets that are positive forthe central probe's detection label and negative for the boundary/distalprobes detection label contain tumor derived viral DNA.

For example, modified primer probe sets 4, 7, and 10 from Table 18 couldbe used for the multiplexed digital PCR reaction to detect tumor-derivedHPV35 DNA, where detection probe 7 is conjugated to FAM and detectionprobes 4 and 10 are conjugated to HEX. Alternatively, detection 7 couldbe conjugated to FAM, probe 4 conjugated to HEX, and probe 10 conjugatedto a different detection moiety.

In some embodiments, the primer/probe sets may be selected so that notwo sets are consecutive in Table 18. For illustration, examples oftriads in this embodiment with no consecutive primer probe sets include{1, 3, 5}, {1, 3, 8}, {4, 6, 9}, . . . {12, 14, 16}. Examples of triadswith a consecutive primer probe set include {1, 2, 5} and {10, 12, 13}.

For the purposes of this application, digital PCR refers to any methodthat will be understood to those versed in the art where PCR reactionsare partitioned into many sub-reactions for analysis. The partitioncould be droplets or microwells or any other partition used for digitalPCR.

It is to be understood that HEX and FAM are used solely for clarity andillustrative purposes in all the examples.

TABLE 18 Primers and probes for HPV35 Amplicon (size) Set (Assay) DetailNamed as Sequence T_(M) ¹ 1 1I (78 bp) Forward primer 1I_Primer15′CTG A+AC G+AC CTT ACA AA31 59.9 (E6 assay) (SEQ ID NO: 233)Reverse primer 1I_Primer2 5′ATA CAC AAT T+CA AA+C +AAA3′ 60.3(SEQ ID NO: 234) TaqMan® probe 1I_Probe5′/Dye/TGA TTT GTG /Quencher-2/CAA CGA 67.5GGT AGA AGA AAG C/Quencher-1 /3′ (SEQ ID NO: 235) 1I_probe_v5′/Dye/CG+A G+G+T +A+GA A+GA 67.4 2 A/Quencher/3′ (SEQ ID NO: 236) 22I (72 bp) Forward primer 2I_Primer1 5′CAA A+CA AGA A+TT AC+A GC3′ 60(E6 assay) (SEQ ID NO: 237) Reverse primer 2I_Primer25′CCT +T+CT +CTA TAT A+CT ATA3′ 59.9 (SEQ ID NO: 238) TaqMan® probe2I_probe 5′/Dye/AG+T +C+A+T ATA +C+CT 67.5 CAC/Quencher/3′(SEQ ID NO: 239) 3 3I (80 bp) Forward primer 3I_Primer15′ATT +C+AA AAA TAA +G+TG AAT3′ 59.7 (E6 assay) (SEQ ID NO: 240)Reverse primer 3I_Primer2 5′TAA CTG TTT GTT +GCA TTG T3′ 59.8(SEQ ID NO: 241) TaqMan® probe 3I_Probe 5′/Dye/TA+G +T+GT GTA +T+G+G68.1 A/Quencher/3′ (SEQ ID NO: 242) 4 4I (75 bp) Forward primer4I_Primer1 5′TGT CAT T+TA +T+TA ATT +AGG T3′ 60 (E6 assay)(SEQ ID NO: 243) Reverse primer 4I_Primer25′TTC TTC TAA +ATG T+CT TTG C3′ 60 (SEQ ID NO: 244) TaqMan® probe4I_Probe 5′/Dye/TG+T +CAA AAA +C+C+G 68.4 C/Quencher/3′ (SEQ ID NO: 245)5 5I (74 bp) Forward primer 5I_Primer1 5′CGA TTC CAT AAC +ATC GGT3′ 60(E6 assay) (SEQ ID NO: 246) Reverse primer 5I_Primer25′TCG GTT TCT CTA CGT GT3′ 59.7 (SEQ ID NO: 247) TaqMan® probe 5I_Probe5′/Dye/CG+G +T+G+T AT+G +TCC 68.1 T/Quencher/3′ (SEQ ID NO: 248) 66I (75 bp) Forward primer 6I_Primer1 5′CAT GGA +GA+A ATA ACT A+CA3′ 60(E7 assay) (SEQ ID NO: 249) Reverse primer 6I_Primer25′CTC ATA ACA +GTA +TA+G GTC3′ 60.2 (SEQ ID NO: 250) TaqMan® probe6I_Probe 5′/Dye/TG+C AA+G A+C+T A+T+G 67.5 T/Quencher/3′(SEQ ID NO: 251) 7 7I (79 bp) Forward primer 7I_Primer15′AAT T+GT GTG ACA GCT CA3′ 60.1 (E7 assay) (SEQ ID NO: 252)Reverse primer 7I_Primer2 5′TAA TTG GAG GTG TCT GGT3′ 60(SEQ ID NO: 253) TaqMan® probe 7I_Probe 5′/Dye/TT+G +C+TT +GTC +C+AG68.1 C/Quencher/3′ (SEQ ID NO: 254) 8 8I (92 bp) Forward primer8I_Primer1 5′TA+A TAT TGT AAC GT+C CTG T3′ 60 (E7 assay)(SEQ ID NO: 255) Reverse primer 8I_Primer25′AT+A A+AT CTT C+CA ATT +TAC G3′ 59.9 (SEQ ID NO: 256) TaqMan® probe8I_Probe 5′/DyeDye/CA+C +TA+C +G+TC TG+T 67.4 G/Quencher/3′(SEQ ID NO: 257) 9 1J (72 bp) Forward primer 1J_Primer15′TGC ATG ATT TGT +GCA AC 3′ 60.1 (E6 assay) (SEQ ID NO: 258)Reverse primer 1J_Primer2 5′CTT G+TT TGC AGT A+TA CAC3′ 60.1(SEQ ID NO: 259) TaqMan® probe 1J_Probe 5′/D ye/AAA G+C+A T+C+C +AT+G68.1 A/Quencher/3′ (SEQ ID NO: 260) 10 2J (95 bp) Forward primer2J_Primer1 5′CAG CGG AGT GAG GTA TAT3′ 60 (E6 assay) (SEQ ID NO: 261)Reverse primer 2J_Primer2 5′AAT T+TT A+AA +CAT TT+C +ATG3′ 60(SEQ ID NO: 262) TaqMan® probe 2J_Probe 5′/DyeDye/AG+A G+AA G+GC C+A+G68 C/Quencher/3′ (SEQ ID NO: 263) 11 3J (76 bp) Forward primer3J_Primer1 5′AAT +ATA +GAT G+GT ATA +G+AT ATA3′ 60 (E6 assay)(SEQ ID NO: 264) Reverse primer 3J_Primer25′AAT A+AA T+GA +CAT AAC T+GT TT3′ 60.1 (SEQ ID NO: 265) TaqMan® probe3J_Probe 5′/DyeDye/TT+C +T+C+C A+TA +CAC 67.8 A/Quencher/3′(SEQ ID NO: 266) 12 4J (75 bp) Forward primer 4J_Primer15′AA+T TA+G GT+G TAT TAC A+TG3′ 59.8 (E6 assay) (SEQ ID NO: 267)Reverse primer 4J_Primer2 5′AAT +CGT TTT +TTT T+CT TCT3′ 60(SEQ ID NO: 268) TaqMan® probe 4J_Probe 5′/DyeDye/C+C+A +G+TT +GAA AA+G67.3 CA/Quencher/3′ (SEQ ID NO: 269) 13 5J (74 bp) Forward primer5J_Primer1 5′CC+A TAA CAT CGG TGG AC3′ 60.1 (E6 assay) (SEQ ID NO: 270)Reverse primer 5J_Primer2 5′AC+A CCT CGG TTT CTC TA3′ 60.1(SEQ ID NO: 271) TaqMan® probe 5J_Probe 5′/DyeDye/TGT +ATG +T+C+C +TG+T67.9 T/Quencher/3′ (SEQ ID NO: 272) 14 6J (77 bp) Forward primer6J_Primer1 5′TTT AGA TTT GGA +ACC CGA3′ 60 (E7 assay) (SEQ ID NO: 273)Reverse primer 6J_Primer2 5′TAT CTT CCT CCT +CCT CT3′ 60.2(SEQ ID NO: 274) TaqMan® probe 6J_Probe 5′/Dye/AC+T +G+A+C +CTA TA+C68.3 T/Quencher/3′ (SEQ ID NO: 275) 15 7J (73 bp) Forward primer7J_Primer1 5′CTA TTG ACG GTC CAG CT3′ 60.5 (E7 assay) (SEQ ID NO: 276)Reverse primer 7J_Primer2 5′CAT TTA C+AA C+AG GAC GTT3′ 60(SEQ ID NO: 277) TaqMan® probe 7J_Probe 5′/Dye/CCA +G+A+C +A+CC 68.7+TCC/Quencher/3′ (SEQ ID NO: 278) 16 8J (75 bp) Forward primer8J_Primer1 5′TGA GGC GAC ACT ACG TC3′ 62.9 (E7 Assay) (SEQ ID NO: 279)Reverse primer 8J_Primer2 5′GTG CCC ATT AAT AAA TCT TCC AA3′ 61.7(SEQ ID NO: 280) TaqMan® probe 8J_Probe5′/Dye/AG+AG+C+ACA+C+ACAT/Quencher/3′ 67.5 (SEQ ID NO: 281)Reverse primer 1I_Primer1 5′CTG A+AC G+AC CTT ACA AA3′ 59.9(SEQ ID NO: 282) TaqMan® probe 1I_Primer2 5′ATA CAC AAT T+CA AA+C +AAA3′60.3 (SEQ ID NO: 283) ¹“Dye” stands for a fluorescent reporter dye. Anyreporter dyes such as FAM™, HEX™, VIC®, Cy5™, and Cy5.5™ that iscompatible with the detection channels for the instrument can be used. Aquencher compatible with the respective dye was used at 3′ end of theprobe (Quencher-1). A second quencher (Quencher-2) such as ZEN wassometimes placed internally between 9^(th) and 10^(th) nucleotide basefrom the reporter dye to enhance the overall dye quenching. ² Theseestimates of melting temperature(T_(M)) were calculated using IDT′Soligo analyzer tools with following settings: (a) For primers: Targettype = DNA, Oligo concentration = 9 μM, Na⁺ concentration = 50 mM, Mg⁺⁺concentration = 3 mM, and dNTPs concentration = 8 mM. (b) for Probe:Target type = DNA, Oligo concentration ³ + = Locked nucleic acid

Example 12: Modified Oligonucleotide Compositions and Methods forDetection of Tumor-Derived HPV45

Provided in Table 19 below are primers and probes for detection oftumor-derived HPV45, where the “+” signifies a locked nucleic acid.Tumor derived HPV45 DNA can be detected and distinguished from non-tumorsources using any three primer/probe sets from Table 19 within a singlemultiplex digital PCR reaction, where the central probe has a detectioncolor distinct from both of the other (boundary) probes. The twoboundary probes may or may not have the same color. The DNA of theprimer/probe sets is modified to include quenchers, dye moieties andlocked nucleic acids. In this example, droplets that are positive forthe central probe's detection label and negative for the boundary/distalprobes detection label contain tumor derived viral DNA.

For example, modified primer probe sets 4, 7, and 10 from Table 19 couldbe used for the multiplexed digital PCR reaction to detect tumor-derivedHPV45 DNA, where detection probe 7 is conjugated to FAM and detectionprobes 4 and 10 are conjugated to HEX. Alternatively, detection 7 couldbe conjugated to FAM, probe 4 conjugated to HEX, and probe 10 conjugatedto a different detection moiety.

In some embodiments, the primer/probe sets may be selected so that notwo sets are consecutive in Table 19. For illustration, examples oftriads in this embodiment with no consecutive primer probe sets include{1, 3, 5}, {1, 3, 8}, {4, 6, 9}, . . . {13, 15, 17}. Examples of triadswith a consecutive primer probe set include {1, 2, 5} and {10, 12, 13}.

For the purposes of this application, digital PCR refers to any methodthat will be understood to those versed in the art where PCR reactionsare partitioned into many sub-reactions for analysis. The partitioncould be droplets or microwells or any other partition used for digitalPCR.

It is to be understood that HEX and FAM are used solely for clarity andillustrative purposes in all the examples.

TABLE 19 Primers and probes for HPV45 Amplicon (size) Set (Assay) DetailNamed as Sequence T_(M) ¹ 1 1K (74 bp) Forward primer 1K_Primer15′CGC TTT GAC GAT CCA AA3′ 60 (E6 assay) (SEQ ID NO: 284) Reverse primer1K_Primer2 5′TCT +TGT A+GT GAT G+TA TTC3′ 60.0 (SEQ ID NO: 285)TaqMan® probe 1K_Probe 5′/Dye/AG+C +TA+C +C+AG +ATT/Quencher/3′ 68.4(SEQ ID NO: 286) 2 2K (85 bp) Forward primer 2K_Primer15′CGT ATC TA+T TGC CTG TGT A3′ 60.4 (E6 assay) (SEQ ID NO: 287)Reverse primer 2K_Primer2 5′CAC TAT A+C+A +TAA AT+C TTT AA3′ 60(SEQ ID NO: 288) TaqMan® probe 2K_Probe5′/Dye/AGC AAC ATT /Quencher-2/GGA ACG 68.4CAC AGA GGT ATA TC/Quencher-1/3′ (SEQ ID NO: 289) 3 3K (74bp)Forward primer 3K_Primer1 5′ATA GAG A+CT +GTA T+AG CA3′ 60 (E6 assay)(SEQ ID NO: 290) Reverse primer 3K_Primer25′ATA T+CT TAA TT+C +T+CT AAT TC3′ 60.1 (SEQ ID NO: 291) TaqMan® probe3K_Probe 5′/Dye/TA+C +ATT TA+T +G+G+C 66.2 AT/Quencher/3′(SEQ ID NO: 292) 4 4K (75 bp) Forward primer 4K_Primer15′TAT T+CA AAC +T+CT GTA +TAT3′ 60 (E6 assay) (SEQ ID NO: 293)Reverse primer 4K_Primer2 5′CAC +C+TT ATT AA+C +AAA TTA T3′ 59.9(SEQ ID NO: 294) TaqMan® probe 4K_Probe5′/Dye/TC+C +AGT G+T+C T+CT C/Quencher/3′ 68.1 (SEQ ID NO: 295) 55K (70 bp) Forward primer 5K_Primer1 5′CCA GA+A ACC +ATT GAA CC3′ 60(E6 assay) (SEQ ID NO: 296) Reverse primer 5K_Primer25′AGC TAT GCT GTG AA+A TCT3′ 60.1 (SEQ ID NO: 297) TaqMan® probe5K_Probe 5′/Dye/CG+T +AG+A +C+AC +CTT/Quencher/3′ 67.4 (SEQ ID NO: 298)6 6K (70 bp) Forward primer 6K_Primer1 5′CGA GGG CAG TGT A+AT AC3′ 60.3(E6 assay) (SEQ ID NO: 299) Reverse primer 6K_Primer25′CTT GTG T+TT CCC TAC GT3′ 60.4 (SEQ ID NO: 300) TaqMan® probe 6K_Probe5′/Dye/CC+T +GG+T +CAC +A+AC 67.5 v1 A/Quencher/3′ (SEQ ID NO: 301)6K_Probe 5′/Dye/TGA CCA GGC /Quencher-2/ACG 68.6 v2GCA AGA AAG A/Quencher-1/3′ (SEQ ID NO: 302) 7 7K (73 bp) Forward primer7K_Primer 5′AAC ACT GCA AGA AAT +TGT A3′ 60.1 (E7 assay) 1(SEQ ID NO: 303) Reverse primer 7K_Primer2 5′CTC GTA ACA CA+A CAG GT3′60.2 (SEQ ID NO: 304) TaqMan® probe 7K_Probe 5′/Dye/TGG AA+C +CT+C A+G+A68.3 A/Quencher/3′ (SEQ ID NO: 305) 8 8K (71 bp) Forward primer8K_Primer1 5′CAA TTA AGC G+AG TCA GAG3′ 60 (E7 assay) (SEQ ID NO: 306)Reverse primer 8K_Primer2 5′GTC GGG CTG GTA GTT GT3′ 58.8(SEQ ID NO: 307) TaqMan® probe 8K_Probe 5′/Dye/CG+A +T+G+A AGC +AG+A 68T/Quencher/3′ (SEQ ID NO: 308) 9 9K (85 bp) Forward primer 9K_Primer15′AAT TTT GT+G +TGT ATG +TTG3′ 59.8 (E7 assay) (SEQ ID NO: 309)Reverse primer 9K_Primer2 5′CTG +CTG TAG T+GT TCT AA3′ 59.7(SEQ ID NO: 310) TaqMan® probe 9K_Probe 5′/Dye/AC+G +G+CA +G+AA +TTG 68A/Quencher/3′ (SEQ ID NO: 311) 10 1L (75 bp) Forward primer 1L_Primer15′CCT ACA AGC +TAC CAG ATT3′ 60.1 (E6 assay) (SEQ ID NO: 312)Reverse primer 1L_Primer2 5′TGC AAT ATA CAC AGG C+AA3′ 59.8(SEQ ID NO: 313) TaqMan® probe 1L_Probe 5′/Dye/CA+C TA+C +AA+G +A+CG67.8 T/Quencher/3′ (SEQ ID NO: 314) 11 2L (75 bp) Forward primer2L_Primer1 5′AAC ATT GGA ACG CAC AG3′ 60.3 (E6 assay) (SEQ ID NO: 315)Reverse primer 2L_Primer2 5′TAT GCT ATA CAG TCT C+TA TAC AC3′ 60.3(SEQ ID NO: 316) TaqMan® probe 2L_Probe 5′/Dye/AG+G +T+AT A+T+C AAT TTG67.8 +CTT/Quencher/3′ (SEQ ID NO: 317) 12 3L (70 bp) Forward primer3L_Primer1 5′CAT +GCC ATA +AAT GTA TAG +AC3′ 60.1 (E6 assay)(SEQ ID NO: 318) Reverse primer 3L_Primer25′CC+A TA+T ACA GA+G TTT GAA T3′ 59.7 (SEQ ID NO: 319) TaqMan® probe3L_Probe 5′/Dye/T+C+C +A+GA ATT A+G+A GA 68.3 (SEQ ID NO: 320) 134L (84 bp) Forward primer 4L_Primer1 5′AAT AAC T+AA TA+C AG+A GTT GT3′60.1 (E6 assay) (SEQ ID NO: 321) Reverse primer 4L_Primer25′TGT CTA CGT TTT T+CT GC3′ 59.9 (SEQ ID NO: 322) TaqMan® probe4L_Probe_v 5′/Dye/AA+C +C+AT T+G+A +ACC 67.9 1 C/Quencher/3′(SEQ ID NO: 323) 4L_Probe_v 5′/Dye/TTG TTA ATA /Quencher-2/AGG 67.7 2TGC CTG CGG TGC/Quencher-1/3′ (SEQ ID NO: 324) 14 5L (91 bp)Forward primer 5L_Primer1 5′TTA AGG A+CA AAC +GAA GAT3′ 60 (E6 assay)(SEQ ID NO: 325) Reverse primer 5L_Primer2 5′AAG TCT TTC TTG CCG TG3′59.6 (SEQ ID NO: 326) TaqMan® probe 5L_Probe_v5′/Dye/TGG ACA GTA /Quencher-2/CCG 68.4 1AGG GCA GTG TAA TA/Quencher-1/3′ (SEQ ID NO: 327) 5L_Probe_v5′/Dye/TAG CTG GAC /Quencher-2/AGT 68.7 2 ACC GAG GGC A/Quencher-1/3′(SEQ ID NO: 328) 15 6L (75 bp) Forward primer 6L_Primer15′TCA GA+A TGA ATT +AGA TCC TG3′ 60.1 (E7 assay) (SEQ ID NO: 329)Reverse primer 6L_Primer2 5′TGC TTC ATC GTT TTC CTC3′ 59.8(SEQ ID NO: 330) TaqMan® probe 6L_Probe5′/Dye/TGA +C+C+T +GTT GT+G T/Quencher/3′ 67.9 (SEQ ID NO: 331) 167L (75 bp) Forward primer 7L_Primer1 5′TAG TCA TGC ACA ACT +ACC3′ 59.7(E7 assay) (SEQ ID NO: 332) Reverse primer 7L_Primer25′TCA CAC TTA CAA CAT A+CA C3′ 59.8 (SEQ ID NO: 333) TaqMan® probe7L_Probe 5′/Dye/CCG A+CG +A+GC CGA/Quencher/3′ 67.5 (SEQ ID NO: 334) 178L (90 bp) Forward primer 8L_Primer1 5′CGG CAG AAT TGA GCT TAC A3′ 62.4(E7 assay) _v1 (SEQ ID NO: 335) 8L_Primer1 5′CGG CAG AAT TGA GCT TAC3′60.4 _v2 (SEQ ID NO: 336) Reverse primer 8L_Primer25′GGA CAC ACA AAG GAC AAG G3′ 62.7 _v1 (SEQ ID NO: 337) 8L_Primer25′GGA CAC ACA AAG GAC AAG3′ 60.2 _v2 (SEQ ID NO: 338) TaqMan® probe8L_Probe_v 5′/Dye/TCG GCA GA+G G+A+C 65.7 1 C/Quencher/3′(SEQ ID NO: 339) 8L_Probe_v 5′/Dye/TCG GC+A GA+G G+A+C 68.3 2C/Quencher/3′ (SEQ ID NO: 340) ¹“Dye” stands for a fluorescent reporterdye. Any reporter dyes such as FAM™, HEX™, VIC®, Cy5™, and Cy5.5™ thatis compatible with the detection channels for the instrument can beused. A quencher compatible with the respective dye was used at 3′ endof the probe (Quencher-1). A second quencher (Quencher-2) such as ZENwas sometimes placed internally between 9^(th) and 10^(th) nucleotidebase from the reporter dye to enhance the overall dye quenching. ² Theseestimates of melting temperature(T_(M)) were calculated using IDT′Soligo analyzer tools with following settings: (a) For primers: Targettype = DNA, Oligo concentration = 9 μM, Na⁺ concentration = 50 mM, Mg⁺⁺concentration = 3 mM, and dNTPs concentration = 8 mM. (b) for Probe:Target type = DNA, Oligo concentration ³ + = Locked nucleic acid

Example 13: Modified Oligonucleotide Compositions and Methods forDetection of Tumor-Derived EBV

Provided in Table 20 below are primers and probes for detection oftumor-derived EBV, where the “+” signifies a locked nucleic acid. Tumorderived EBV DNA can be detected and distinguished from non-tumor sourcesusing any three consecutive primer/probe sets from Table 20 within asingle multiplex digital PCR reaction, where the central probe has adetection color distinct from both of the other (boundary) probes. Thetwo boundary probes may or may not have the same color. In this example,droplets that are positive for the central probe's detection label andnegative for the boundary/distal probes detection label contain tumorderived viral DNA.

For example, modified primer probe sets 4, 5, and 6 from Table 20 couldbe used for the multiplexed digital PCR reaction to detect tumor-derivedEBV DNA, where detection probe 5 is conjugated to FAM and detectionprobes 4 and 6 are conjugated to HEX. Alternatively, detection 5 couldbe conjugated to FAM, probe 4 conjugated to HEX, and probe 6 conjugatedto a different detection moiety.

For illustration, examples of three consecutive primer probe setsinclude {1, 2, 3}, {2, 3, 4}, {3, 4, 5}, . . . {15, 16, 17}.

For the purposes of this application, digital PCR refers to any methodthat will be understood to those versed in the art where PCR reactionsare partitioned into many sub-reactions for analysis. The partitioncould be droplets or microwells or any other partition used for digitalPCR.

It is to be understood that HEX and FAM are used solely for clarity andillustrative purposes in all the examples.

TABLE 20 Primers and probes for Epstein-Barr virus (EBV) Amplicon (size)Set (Assay) Detail Named as Sequence T_(M) ¹ 1 1M (70 bp) Forward primer1M_Primer1 5′GGG AGA CCG AAG TGA A3′ 59.9 (SEQ ID NO: 341)Reverse primer 1M_Primer2 5′TCC ACT TAC CTC TGG C3′ 59.7(SEQ ID NO: 342) TaqMan® probe 1M_Probe5′/Dye/CT+G GA+C C+A+A +CCC G/Quencher/3′ 68 (SEQ ID NO: 343) 22M (80 bp) Forward primer 2M_Primer1 5′CTC GGA CAG CTC CTA AG3′ 60.3(SEQ ID NO: 344) Reverse primer 2M_Primer2 5′ACC CTT CT+A CGG ACT C3′60.2 (SEQ ID NO: 345) TaqMan® probe 2M_Probe5′/Dye/CG+C CCA GT+C +CT+A C/Quencher/3′ 68.5 (SEQ ID NO: 346) 33M (87 bp) Forward primer 3M_Primer1 5′TCT CTT AGA GAG +TGG CT3′ 60.8(SEQ ID NO: 347) Reverse primer 3M_Primer25′TTC TTC +TAT G+TA GAC +AGA3′ 60.4 (SEQ ID NO: 348) TaqMan® probe3M_Probe 5′/Dye/CG+C +ATT A+G+A +G+AC 68.5 C/Quencher/3′(SEQ ID NO: 349) 4 4M (78 bp) Forward primer 4M_Primer15′TGC CTA C+AT +TCT +ATC TTG3′ 59.9 (SEQ ID NO: 350) Reverse primer4M_Primer2 5′ACG GGT TTC CAA GAC TA3′ 59.8 (SEQ ID NO: 351)TaqMan® probe 4M_Probe 5′/Dye/TA+T +G+TT TG+T +CC+C 69.1 C/Quencher/3′(SEQ ID NO: 352) 5 5M (75 bp) Forward primer 5M_Primer15′CAC TCT CAG TA+A TTC CCT C3′ 60.2 (SEQ ID NO: 353) Reverse primer5M_Primer2 5′CAA AGA +TT+G TTA GTG G+AA T3′ 60 (SEQ ID NO: 354)TaqMan® probe 5M_Probe 5′/Dye/TCC +CTA +CC+A +GG+A A/Quencher/3′ 68.6(SEQ ID NO: 355) 6 6M (76 bp) Forward primer 6M_Primer15′CCG CTA GGA TAT GAC GT3′ 59.9 (SEQ ID NO: 356) Reverse primer6M_Primer2 5′TTA CAA TAT +AAT TA+G +C+CA3′ 59.9 (SEQ ID NO: 357)TaqMan® probe 5M_Probe 5′/Dye/ACC TCT AGC /Quencher-2/ATC TGC 68.2TAT GCG AAT GC/Quencher-1/3′ (SEQ ID NO: 358) 7 17M (81 Forward primer7M_Primer1 5′GCT TAC CCC TCC ACA A3′ 60.4 bp) (SEQ ID NO: 359)Reverse primer 7M_Primer2 5′GGC ACC GTT AGT GTT G3′ 59.7(SEQ ID NO: 360) TaqMan® probe 7M_Probe5′/Dye/TAC CCC TCC /Quencher-2/TAC CCC 68.5 TCT GCC/Quencher-1/3′(SEQ ID NO: 361) 8 8M (91 bp) Forward primer 8M_Primer15′TAC CGA ACT TCA ACC CA3′ 60.1 (SEQ ID NO: 362) Reverse primer8M_Primer2 5′TTG +ATG AGT AAG AGG GTG3′ 59.9 (SEQ ID NO: 363)TaqMan® probe 8M_Probe 5′/Dye/CC+A TCA C+CA +C+C+A 68.9 v1 C/Quencher/3′(SEQ ID NO: 364) 8M_Probe 5′/Dye/TG+C +C+AG A+CC AAT 68.4 v2C/Quencher/3′ (SEQ ID NO: 365) 9 9M (75 bp) Forward primer 9M_Primer15′AGC ACC CCA +AAT GAT C3′ 60.2 (SEQ ID NO: 366) Reverse primer9M_Primer2 5′TAA TGG +CAT AGG TGG AA3′ 59.8 (SEQ ID NO: 367)TaqMan® probe 9M_Probe 5′/Dye/TCC AGA ACC /Quencher-2/ACG GTC 68.1CCC G/Quencher-1/3′ (SEQ ID NO: 368) 10 10M (87 Forward primer10M_Primer 5′ATC AAC GAC CAA CAA +TTA C3′ 60 bp) 1 (SEQ ID NO: 369)Reverse primer 10M_Primer 5′TTG AGT CTT AGA GGG T+TG3′ 60.1 2(SEQ ID NO: 370) TaqMan® probe 10M_Probe5′/Dye/CC+C +GAG +GG+T AG+A T/Quencher/3′ 68.8 (SEQ ID NO: 371) 1111M (76 Forward primer 11M_Primer 5′TTG GAG ACC AGA GCC3′ 59.6 bp) 1(SEQ ID NO: 372) Reverse primer 11M_Primer 5′TTG TCC CTG ATG AAG +AC3′60.1 2 (SEQ ID NO: 373) TaqMan® probe 11M_Probe5′/Dye/GC+C +T+GA A+C+T AA+G 67.8 T/Quencher/3′ (SEQ ID NO: 374) 1212M (86 Forward primer 12M_Primer 5′ACT CAC CAA CTC CTG G3′ 60 bp) 1(SEQ ID NO: 375) Reverse primer 12M_Primer5′TTC ATG TAT TG+G TGA AAC G3′ 60.1 2 (SEQ ID NO: 376) TaqMan® probe12M_Probe 5′/Dye/CAA TGC CGC /Quencher-2/CCC CGT 68TTG TAG/Quencher-1/3′ (SEQ ID NO: 377) 13 13M (78 Forward primer13M_Primer 5′CGG AGT CCC ATA +ATA GC3′ 59.9 bp) 1 (SEQ ID NO: 378)Reverse primer 13M_Primer 5′GTC TGC GGG GTC TAT AG3′ 60.1 2(SEQ ID NO: 379) TaqMan® probe 13M_Probe5′/Dye/CA+G +AG+G +C+TC CCA T/Quencher/3′ 68.7 (SEQ ID NO: 380) 1414M (84 Forward primer 14M_Primer 5′AAA GTT GG+G ATT A+CA TTT3′ 59.9 bp)1 (SEQ ID NO: 381) Reverse primer 14M_Primer 5′GGC GAG GTC TTT T+AC TG3′60.4 2 (SEQ ID NO: 382) TaqMan® probe 14M_Probe5′/Dye/TGA GAC AAC /Quencher-2/AGA ATC 68TCC TAG CTC AGA TGA/Quencher-1/3′ (SEQ ID NO: 383) 15 15M (86Forward primer 15M_Primer 5TAGGCCCACTTAACACTAC3′ 60.6 bp) 1(SEQ ID NO: 384) Reverse primer 15M_Primer 5′GGAGGCCCTTAGACTTAC3′ 60.5 2(SEQ ID NO: 385) TaqMan® probe 15M_Probe5′/Dye/CGCCTCTCCATTCATCATGTAACCCAC/ 68.4 Quencher/3′ (SEQ ID NO: 386) 1616M (85 Forward primer 16M_Primer 5′ATCCCTAGGATAATACCACAC3′ 60.5 bp) 1(SEQ ID NO: 387) Reverse primer 16M_Primer 5′CACGTAAAGCCACAAGC3′ 60.8 2(SEQ ID NO: 388) TaqMan® probe 16M_Probe5′/Dye/AGCAGTGTAGTCCTGTCAATCTCCTGA/ 68.3 Quencher/3′ (SEQ ID NO: 389) 171N (93 bp) Forward primer 1N_Primer1 5′CTC CTA AGA AGG +CAC C3′ 60.4(SEQ ID NO: 390) Reverse primer 1N_Primer2 5′CTC CTC TTC TTG CTG GA3′60.1 (SEQ ID NO: 391) TaqMan® probe 1N_Probe5′/Dye/CA+A +G+AA C+C+C AG+A 68.9 C/Quencher/3′ (SEQ ID NO: 392) 182N (81 bp) Forward primer 2N_Primer1 5′ATT +AGA GAC C+AC TTT +GAG3′ 60.1(SEQ ID NO: 393) Reverse primer 2N_Primer25′TTA GTC +TTC ATC CTC T+TC T3′ 60.3 (SEQ ID NO: 394) TaqMan® probe2N_Probe 5′/Dye/CG+C C+AA T+C+T +G+TC 68.7 T/Quencher/3′(SEQ ID NO: 395) 19 3N (83 bp) Forward primer 3N_Primer15′TCT AAT TGT TGA CAC +GGA T3′ 60.3 (SEQ ID NO: 396) Reverse primer3N_Primer2 5′TGT CT+G ACA GTT GTT CC3′ 60.4 (SEQ ID NO: 397)TaqMan® probe 3N_Probe 5′/Dye/CTT GGA AAC /Quencher-2/CCG TCA 68.1CTC TCA GTA ATT CC/Quencher-1/3′ (SEQ ID NO: 398) 20 4N (84 bp)Forward primer 4N_Primer1 5′TCA CCT CTT GAT AGG GAT C3′ 59.8(SEQ ID NO: 399) Reverse primer 4N_Primer2 5′ATT AGC CAT CCA AAG C+AT3′60.3 (SEQ ID NO: 400) TaqMan® probe 4N_Probe5′/Dye/CGG GCA TGG /Quencher-2/ACC TCT 67.7 AGC ATC T/Quencher-1/3′(SEQ ID NO: 401) 21 5N (89 bp) Forward primer 5N_Primer15′ATA TTG +TAA GA+C A+A+T CAC3′ 60.3 (SEQ ID NO: 402) Reverse primer5N_Primer2 5′ATG TGG CTG GAC CAA3′ 60.1 (SEQ ID NO: 403) TaqMan® probe5N_Probe 5′/Dye/CC+T G+AG +GGG C+TG T/Quencher/3′ 68.4 (SEQ ID NO: 404)22 6N (84 bp) Forward primer 6N_Primer1 5′CTC TAC GCC CGA CAG3′ 60.2(SEQ ID NO: 405) Reverse primer 6N_Primer2 5′TTG GTG GCA TC+A TGA G3′59.7 (SEQ ID NO: 406) TaqMan® probe 6N_Probe5′/Dye/TGT CAC CTC /Quencher-2/TGT CAC 67.7 AAC CGA GG/Quencher-1/3′(SEQ ID NO: 407) 23 7N (98 bp) Forward primer 7N_Primer15′ACC CAC ACC ACT ACT C3′ 59.6 (SEQ ID NO: 408) Reverse primer7N_Primer2 5′TCT GGC ACA TGC AAG +A3′ 60.4 (SEQ ID NO: 409)TaqMan® probe 7N_Probe 5′/Dye/TAC CGA ACT /Quencher-2/TCA ACC 68.2CAC ACC ATC AC/Quencher-1/3′ (SEQ ID NO: 410) 24 8N (86 bp)Forward primer 8N_Primer1 5′AAT CAA TGC ACC CTC +TTA3′ 59.9(SEQ ID NO: 411) Reverse primer 8N_Primer25′AAT G+TT ATA AAA TAC AG+T +CG3′ 60.2 (SEQ ID NO: 412) TaqMan® probe8N_Probe 5′/Dye/TGA TCC AGA /Quencher-2/TAG TCC 68.4AGA ACC ACG GT/Quencher-1/3′ (SEQ ID NO: 413) 25 9N (85 bp)Forward primer 9N_Primer1 5′ATT ACC CCC CTC ACA AT3′ 59.8(SEQ ID NO: 414) Reverse primer 9N_Primer25′TAG ATG AT+G TAA TTG TT+G GT3′ 59.9 (SEQ ID NO: 415) TaqMan® probe9N_Probe 5′/Dye/CAC CAC CAG /Quencher-2/CAG CAC 68.1 CAG C/Quencher-1/3′(SEQ ID NO: 416) 26 10N (82 Forward primer 10N_Primer5′ACA AGC AAC GCA AGC3′ 60.5 bp) 1 (SEQ ID NO: 417) Reverse primer10N_Primer 5′AGG ACT GGA C+TT AGT TCA3′ 60.7 2 (SEQ ID NO: 418)TaqMan® probe 10N_Probe 5′/Dye/ACC TTG GAG /Quencher-2/ACC AGA 68GCC AAA CA/Quencher-1/3′ (SEQ ID NO: 419) 27 11N (75 Forward primer11N_Primer 5′CGT +TTG TAG AAA T+TC ACA C3′ 60.2 bp) 1 (SEQ ID NO: 420)Reverse primer 11N_Primer 5′TCT GGG CTA TT+A TGG GA3′ 60.1 2(SEQ ID NO: 421) TaqMan® probe 11N_Probe 5′/Dye/TCA +C+C+A ATA +C+AT 68+GA/Quencher/3′ (SEQ ID NO: 422) 28 12N (77 Forward primer 12N_Primer5′CCA TTC TCT TCC CCG AT3′ 60.3 bp) 1 (SEQ ID NO: 423) Reverse primer12N_Primer 5′AAA TGT AA+T CCC AA+C TTT3′ 60.3 2 (SEQ ID NO: 424)TaqMan® probe 12N_Probe 5′/Dye/CC+G +C+A+G ACT +TA+G 67.7 A/Quencher/3′(SEQ ID NO: 425) ¹“Dye” stands for a fluorescent reporter dye. Anyreporter dyes such as FAM™, HEX™, VIC®, Cy5™, and Cy5.5™ that iscompatible with the detection channels for the instrument can be used. Aquencher compatible with the respective dye was used at 3′ end of theprobe (Quencher-1). A second quencher (Quencher-2) such as ZEN wassometimes placed internally between 9^(th) and 10^(th) nucleotide basefrom the reporter dye to enhance the overall dye quenching. ² Theseestimates of melting temperature(T_(M)) were calculated using IDT′Soligo analyzer tools with following settings: (a) For primers: Targettype = DNA, Oligo concentration = 9 μM, Na⁺ concentration = 50 mM, Mg⁺⁺concentration = 3 mM, and dNTPs concentration = 8 mM. (b) for Probe:Target type = DNA, Oligo concentration ³ + = Locked nucleic acid

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-20. (canceled)
 21. A composition for detecting tumor-derived HumanPapilloma Virus type 18 (HPV18) in a sample from a subject, comprisingat least a triad of modified oligonucleotide primer/probe sets selectedfrom the group consisting of: Set 1: SEQ ID NO:80, SEQ ID NO:81, and SEQID NO:82, Set 2: SEQ ID NO:83, SEQ ID NO:84, and SEQ ID NO:85, Set 3:SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO:88, Set 4: SEQ ID NO:89, SEQID NO:90, and SEQ ID NO:91, Set 5: SEQ ID NO:92, SEQ ID NO:93, and SEQID NO:94, Set 6: SEQ ID NO:95, SEQ ID NO:96, and SEQ ID NO:97, Set 7:SEQ ID NO:98, SEQ ID NO:99, and SEQ ID NO:100, Set 8: SEQ ID NO:101, SEQID NO:102, and SEQ ID NO:103, Set 9: SEQ ID NO:104, SEQ ID NO:105, andSEQ ID NO:106, Set 10: SEQ ID NO:107, SEQ ID NO:108, and SEQ ID NO:109,Set 11: SEQ ID NO:110, SEQ ID NO:111, and SEQ ID NO:112, Set 12: SEQ IDNO:113, SEQ ID NO:114, and SEQ ID NO:115, Set 13: SEQ ID NO:116, SEQ IDNO:117, and SEQ ID NO:118, Set 14: SEQ ID NO:119, SEQ ID NO:120, and SEQID NO:121, Set 15: SEQ ID NO:122, SEQ ID NO:123, and SEQ ID NO:124, Set16: SEQ ID NO:125, SEQ ID NO:126, and SEQ ID NO:127, Set 17: SEQ IDNO:128, SEQ ID NO:129, and SEQ ID NO:130, and Set 18: SEQ ID NO:131, SEQID NO:132, and SEQ ID NO:133, wherein a first primer/probe set of thetriad is configured to produce a first amplicon signal, wherein a secondprimer/probe set of the triad is configured to produce a second ampliconsignal, wherein a third primer/probe set of the triad is configured toproduce a third amplicon signal, and wherein the primer/probe set of thetriad with the smallest set number corresponds to the first primer/probeset and the primer/probe set of the triad with the largest set numbercorresponds to the third primer/probe set.
 22. The composition of claim21, wherein the triad contains three primer/probe sets of which no twoprimer/probe sets are consecutive.
 23. The composition of claim 21,wherein the triad contains three non-consecutive primer/probe sets. 24.The composition of claim 21, comprising primer/probe sets 4, 7, and 10.25. The composition of claim 24, wherein detection probe 7 is conjugatedto reporter moiety FAM and detection probes 4 and 10 are conjugated toreporter moiety HEX.
 26. The composition of claim 24, wherein detectionprobe 7 is conjugated to reporter moiety FAM, detection probe 4 isconjugated to reporter moiety HEX, and detection probe 10 is conjugatedto reporter moiety Cy5™ or reporter moiety Cy5.5.
 27. The composition ofclaim 21, comprising primer/probe sets 1, 3, and 5, or primer/probe sets1, 3, and
 8. 28. The composition of claim 21, comprising primer/probesets 4, 6, and
 9. 29. The composition of claim 21, comprisingprimer/probe sets 14, 16, and
 18. 30. The composition of claim 21,comprising primer/probe sets 1, 2, and
 5. 31. The composition of claim21, comprising primer/probe sets 10, 12, and
 13. 32. The composition ofclaim 21, further comprising a reporter moiety.
 33. The composition ofclaim 32, wherein the reporter moiety comprises a reporter dye.
 34. Thecomposition of claim 33, wherein the reporter dye comprises FAM, HEX,VIC, Cy5™, or Cy5.5.
 35. A method for detecting tumor-derived HumanPapilloma Virus type 16 (HPV18) in a sample from a subject, the methodcomprising: providing at least a triad of modified oligonucleotideprimer/probe sets of the composition of claim 21; fractionating aplurality of HPV DNA fragments from the sample into droplets at aconcentration wherein only 0 or 1 molecule of the DNA fragments ispresent in each droplet; amplifying HPV DNA in each droplet with thetriad of primer/probe sets to produce amplicon signals; and detecting ineach droplet any amplicon signals; wherein detection within a droplet ofthe second amplicon, but not the first or third amplicon, indicates thatthe HPV DNA fragment fractionated into the droplet is a tumor-derivedHPV DNA fragment.
 36. The method of claim 35, wherein the DNA fragmentsare fractionated into micro-droplets by emulsification.
 37. The methodof claim 35, wherein the DNA is amplified using a PCR based method. 38.The method of claim 35, wherein the sample is a blood, saliva, gargle,or urine sample.
 39. The method of claim 38, wherein the sample is ablood sample.